RU2511418C2 - Patient-specific stem cell lines derived from human parthenogenetic blastocysts - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. described are methods for generating HLA homozygous parthenogenetic human stem cell (hpSC-Hhom) lines from both HLA homozygous and HLA heterozygous donors. These hpSC-Hhom lines demonstrate typical human embryonic stem cell morphology, expressing appropriate stem cell markers and possessing high levels of alkaline phosphatase and telomerase activity. Additionally, injection of these cell lines into immunodeficient animals leads to teratoma formation. Furthermore, in the case of HLA heterozygous donors, the hpSC-Hhom lines inherit the haplotype from only one of the donor's parents. SNP data analysis suggests that hpSC-Hhom lines derived from HLA heterozygous oocyte donors are homozygous throughout the genome as assessed by single-nucleotide polymorphism (SNP) analysis.

EFFECT: described protocol minimises the use of animal-derived components, which makes the stem cells more suitable for clinical application.

13 cl, 11 dwg, 15 tbl, 2 ex

Description

BACKGROUND

FIELD OF THE INVENTION

The present invention relates mainly to embryonic stem cells and, more specifically, to a method for producing human HLA-homozygous parthenogenetic human stem cell lines for cell therapy.

BACKGROUND

The first human embryonic stem cells (ESC) were obtained from the internal cell mass (ICM) of a blastocyst derived from a fertilized oocyte capable of infinite division and differentiation into cells of all cell types. Embryonic stem cell is a potentially limitless source of pluripotent cells for transplant cell therapies.

Human embryonic stem cells (ES) are pluripotent cells that can differentiate into a wide variety of cell types. After injection, mice with immunodeficiency embryonic stem cells form differentiated tumors (teratomas). However, embryonic stem cells that are induced in vitro to form embryoid bodies (EBs) provide a source of embryonic stem cell lines that are susceptible to differentiation into numerous cell types characteristic of several tissues under certain growth conditions. For example, ES cells differentiate into neurons in the presence of nerve growth factor and retinoic acid.

Human embryonic stem cells have the potential to have a significant beneficial effect on patients, provided that the problem of immune rejection can be solved. Embryonic stem cells that are genetically related to the recipient can overcome these rejection problems. Currently, human embryonic stem cells (hES) are obtained from three sources: blastocysts remaining after infertility treatment and provided for research, blastocysts obtained from donor gametes (oocytes and sperm), and nuclear transfer products (NT). Cadaveric fetal tissue is the only source of human embryonic germ cells (hEG). HES and hEG cells offer remarkable scientific and therapeutic possibilities, including the ability to generate more specialized cells or tissues. However, ethical considerations regarding the sources of hES and hEG cells, and fears that using NT for research could lead to the use of NT to create humans, have sparked much debate and debate in society.

Parthenogenetic activation of mammalian oocytes can be used as an alternative to sperm fertilization / NT to obtain oocytes for the generation of embryonic stem cells. Parthenogenetic activation is the production of embryonic cells, with a finite development in an adult or without it, from a female gamete in the absence of any contribution from the male gamete.

Parthenogenetic activation of oocytes is a relatively simple way to create histocompatible stem cells compared to SCNT, because it does not require the use of sophisticated equipment necessary for microscopic manipulation of the oocyte. Parthenogenetic stem cells are formed from unfertilized oocytes and contain genetic material exclusively from the oocyte donor (potential patient). In addition, after targeted cell differentiation, autologous cells can be transplanted without fear of a threat of immune rejection. Parthenogenetic murine MHC-homozygous stem cell lines and one parthenogenetic heterozygous embryonic primate stem cell line (Cyno-1) have already been obtained, and cell pluripotency has been demonstrated in these lines.

As mentioned above, the greatest risk after transplantation of allogeneic tissue and organ is immune rejection. The degree of risk is proportional to the degree of incompatibility between the antigen-presenting proteins of the cell surface of the donor and recipient. In an ideal graft, donor tissue is histocompatible with the recipient in the major histocompatibility complex (MHC). The human leukocyte antigen system (HLA) is a nomenclature that characterizes human MHC and represents antigens important for transplantation. HLA antigen-matched tissue of the donor and the recipient reduces the likelihood of a cytotoxic T-cell response in the recipient's body and, thus, significantly increases the likelihood of transplant survival.

HLA haplotypes of MHC class I and II are specific sets of alleles of the HLA-A, -B, -DR loci inherited together from the parent. Despite the high degree of HLA polymorphism, there are only 200 common HLA haplotypes in the US white population. The indicated diversity of HLA in combination with the coefficient of heterozygous selection means that the probability of detecting the coincidence of the donor-recipient is in the range from one in 1000 to one in several million, due to the uniqueness of the tissue type due to the combination of these allelic variants in a heterozygous individual.

Transplant-based stem cell therapy faces the same HLA compatibility issues that limit the use of solid allogeneic organ transplants due to immune rejection. HLA-compatible stem cell lines can overcome the risk of immune rejection. To obtain parthenogenetic cells, HLA-heterozygous cell lines are obtained from HLA-heterozygous donors by activation of oocytes using a combination of A23187 and 6-DMAP. Since these cells are HLA-compatible with an oocyte donor, their ability to provide tissue-compatible derivatives is limited.

MHC compatibility between donor and recipient is significantly increased if the donor cells are HLA homozygous, i.e. contain identical alleles for each antigen-presenting protein. In addition, if homozygous donor cells have a haplotype that is often found in a population, these cells can be used for treatment using stem cell transplantation for a large number of individuals.

SUMMARY OF THE INVENTION

The present invention relates to methods for producing human HLA-homozygous parthenogenetic stem cell lines (hpSC-Hhom) from both HLA-homozygous and HLA-heterozygous donors. These hpSC-Hhom lines demonstrate the morphology of human embryonic stem cells, expressing typical stem cell markers (i.e., SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and OST-4) and having high levels alkaline phosphatase and telomerase activity. In addition, the injection of these cell lines to animals with immunodeficiency leads to the formation of teratoma. Analysis of SNP data shows that the hpSC-Hhom lines obtained from HLA heterozygous oocyte donors are homozygous throughout the genome. The described protocol minimizes the use of components derived from animals, which makes these stem cells ideally suited for clinical use.

In one embodiment, the present invention describes an isolated human stem cell line derived from parthenogenetic blastocysts in which at least one cell comprising the cell line is heterozygous for one or more single nucleotide polymorphisms (SNPs) homozygous for one or more HLA alleles or includes a combination of homozygous and heterozygous SNPs.

In one aspect, at least one cell is HLA allele homozygous. In another aspect, at least one cell forms a teratoma after transplantation of a immunocompromised mouse. In another aspect, at least one cell is MHC compatible with a blastocyst donor.

In a related aspect, at least one cell is MHC compatible with a direct relative of the blastocyst donor, including that at least one cell has been genetically imprinted to a significant extent in accordance with the origin of the donor.

In one aspect, at least one cell (i) will proliferate in an in vitro culture for more than one year, (ii) retain the potential for differentiation into derivatives of endoderm, mesoderm, and ectoderm tissues throughout the culture and (iii) amenable to inhibition of differentiation when it cultured in the feeding sublayer of fibroblasts.

In another aspect, at least one cell differentiates into a cell, including a neuronal cell, a heart cell, a smooth muscle cell, striated muscle cells, an endothelial cell, an osteoblast, an oligodendrocyte, a hematopoietic cell, a fat cell, a stromal cell, chondrocyte, astrocytitis, kera a pancreatic islet cell, a lymphoid progenitor cell, a mast cell, a mesoderm cell, and an endoderm cell. In a related aspect, at least one cell does not have the ability to form a viable organism.

In another embodiment, the invention provides a method of treating a subject in need thereof, comprising administering a cell composition comprising differentiated cells, wherein the differentiated cells are derived from a stem cell line derived from parthenogenetic blastocysts, where at least one cell constituting the cell line, is heterozygous for one or more single nucleotide polymorphisms (SNPs), homozygous for one or more HLA alleles, or includes a combination of homozygous and heteroses Igotny SNP.

In one aspect, the subject has a disease, including Parkinson’s disease, Huntington’s disease, Alzheimer's disease, ALS, defects or injuries of the spinal cord, multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, macular degeneration, defects or damage to cartilage, burns, foot ulcers, vascular disease, urinary tract disease, AIDS and cancer.

In one embodiment of the present invention, a stem cell library is described comprising autologous or allogeneic stem cells, in which the stem cells are derived from parthenogenetically activated oocytes from one or more human donors and where the stem cells are HLA-homozygous stem cells. In one aspect, each member of the library is identified as complete siblings, half-siblings or unrelated according to markers of single nucleotide polymorphism (SNP). In another aspect, each member of the library is HLA-type, and the member is HLA-compatible with potential recipients for therapeutic use.

In another aspect, the oocyte donor is histocompatible with a member of the library. In a related aspect, the library member has undergone genomic imprinting according to the origin of the oocyte donor, including that the stem cells of the library can be obtained from an HLA heterozygous oocyte donor. In yet another aspect, each member of the library is homozygous for a combination of MHC / HLA alleles that is different from other members of the library.

In one aspect, each member of the library is at least homozygous for one or more class I HLA genes and class II HLA genes. In a related aspect, class I HLA genes include combinations of the HLA A *, HLA B *, HLA and Cw * haplotypes. In another related aspect, class II HLA genes include combinations of the HLA haplotypes DRB1 *, DRB3 *, DRB4 *, DRB5 *, DQA1 * and DQB1 *.

In another embodiment of the present invention, a method for generating HLA-homozygous stem cells is described, comprising screening oocyte donors for HLA haplotypes widespread in this population group, incubating human oocytes in metaphase II, in vitro fertilization (IVF), incubating cells in IVF environments, including ionophore, incubation of cells in IVF environments, including puromycin, and incubation of cells in fresh IVF, where one or more incubations are carried out at different I mean the partial pressure of O ₂ and where the internal cell masses (ICM) obtained from the cells produce cultured stem cells.

Examples of methods and compositions of the present invention are described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows specific markers characteristic of phESC lines. Undifferentiated phESC colonies on the human cell sublayer (A-F), negative staining for SSEA-1 (G-L), expression of cell surface markers SSEA-3 (M-R), SSEA-4 (S-X). Increase (A) by (E) × 100; (F) × 200; (G) by (X) × 400. Positive alkaline phosphatase staining of phESC colonies on feeder cells (A-F), OCT-4 (G-L), TRA-1-60 (K-R) and TRA-1-81 (S-X). Magnification (A, B, O, R) × 100; (C-F, M, S, X) × 200; (G-L, N, P, Q, T-W) × 400.

Figure 2 shows that phESC show a high level of telomerase activity compared with positive control cells: "+" - extract of 500 cells; “-” - cell extract after heat treatment with inactivated telomerase; “Control +” - telomerase-positive cell extract (TRAPEZE kit was used); “B” - CHAPS lysis buffer, control of primer-dimer / PCR contamination; TSR8 - matrix for quantitative control of telomerase (0.1 and 0.2 amol / μl); "M" - marker, DNA ladder.

Figure 3 shows the karyotyping of phESC lines on the Giemsa disk. phESC-1 (A), phESC-3 (B), phESC-4 (C), phESC-5 (D) and phESC-6 (E) lines have a normal 46, XX karyotype. The phESC-7 line has karyotype 47, XXX (F).

Figure 4 shows in vitro differentiation of phESC into derivatives of all three germ layers. The differentiation of the ectoderm is represented by positive immunocytochemical staining for neuron-specific markers neurofilament 68 (A), NCAM (B), tubulin beta III (C) and glial cell marker GFAP (D, M). Differentiated cells were positive for mesoderm markers: muscle-specific alpha-actinin (G) and desmin (J), endothelial markers PECAM-1 (E) and VE-cadherin (F). Endoderm differentiation is represented by positive staining for alpha-fetoprotein (H, L). phESC produce pigmented epithelium-like cells (I, K). Magnification (I) × 100; (A-H, J-M) × 400.

Figure 5 shows in vivo differentiation of phESC and teratoma formation in SCID mice. Immunofluorescence staining for markers of all three germ layers. Muscle actin, a marker of mesoderm cells, is located around other components and is clearly identified (A). The presence of fibronectin in higher amounts is specific for connective tissue of mesodermal origin (B). The areas of neural differentiated cells (of ectodermal origin) are extensive and intensively labeled with antibodies against beta-tubulin (C). Alpha-fetoprotein, a marker of immature endodermal cells, can be extracted from areas of glandular appearance (D). The nuclei were stained with DAPI - (A), (D). Magnification (A), (C), (D) × 100; (B) × 200.

Figure 6 shows the analysis of RT-PCR expression of imprinted genes. Two hESC, hESC1, and hESC2 lines from unused IVF embryos were used as positive controls for analysis of expression of paternal expressed genes SNRPN and PEG1_2 and maternal expressed genes TSSC5 and H19 in phESC lines: phESC-1; phESC-3; phESC-4; phESC-5; phESC-6 and phESC-7 (1, 3, 4, 5, 6, and 7, respectively). The PEG1_1 gene is expressed biallelic and was used as an additional control. GAPDH was included as a quantitative control of mRNA. RT data show the absence of genomic contamination in RT samples.

Figure 7 shows specific markers characteristic of hpSC-Hhom lines. Cells from all four hpSC-Hhom lines show positive staining for SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT-4 (nuclei stained using DAPI), alkaline phosphatase and negative staining for SSEA -one. Mouse embryonic stem cells (ESC) were used as a positive control for staining for SSEA-1.

Figure 8 shows that the hpSC-Hhom lines show high levels of telomerase activity compared to positive control cells: “K1 +” is an extract of telomerase-positive cells (used the TRAPEZE kit); "K2 +" - an extract of 500 cells; “K-” or “-” - cell extract after heat treatment with inactivated telomerase; Buf — CHAPS lysis buffer, control of primer-dimer / PCR contamination; TSR - matrix for quantitative control of telomerase (0.1 and 0.2 amol / μl); "M" - marker, DNA ladder.

Figure 9 shows Giemsa disk karyotyping of HLA-homozygous parthenogenetic human stem cell lines: hpSC-Hhom-1 (A) and hpSC-Hhom-4 (B) lines have a normal 46, XX karyotype; hpSC-Hhom-2 (C) line has 15% of cells with karyotype 47, XX, +8 karyotype - aneuploidy of chromosome 8; The hpSC-Hhom-3 (D) line has 4.2% of cells with 47, XX, +1 karyotype - chromosome 1 aneuploidy.

Figure 10 shows in vitro differentiation of hpSC-Hhom-4 into derivatives of all three germ layers: ectoderm differentiation is represented by positive immunocytochemical staining for neuron-specific markers neurofilament 68 (A) and NCAM (B); differentiation of the endoderm is represented by positive staining for alpha-fetoprotein (C); differentiated cells were positive for mesoderm markers specific for the muscles of desmin (D) and alpha-actinin (E); magnification × 200 (AE).

Figure 11 shows in vivo differentiation of hpSC-Hhom-4. Teratoma formation in SCID mice. Derivatives of all three embryonic germ layers (ectoderm, endoderm, and mesoderm): well-formed respiratory-type glands surrounded by mesenchymal cells, hematoxylin-eosin staining (h / e), magnification × 140 (A); possibly a neural tube with a single layer of cells; on the right is the endodermal gland and below the cartilage differentiation, h / e staining, magnification × 70 (B); a bone surrounded by mesenchymal cells, in the center is a tubular gland with cubic epithelium, picrofuscin staining, magnification × 140 (C); well-formed bone surrounded by mesenchymal cells and adipose tissue, h / e staining, magnification × 140 (D); left endodermal glands, mesodermal and adipose tissue, collagen; the bone is visible on the right and bottom, staining according to Kraberg, magnification × 70 (E); endodermal glandular structure surrounded by mesenchymal cells, high production of collagen fibers, Van Gieson staining, magnification × 280 (F); stratified epithelium, hyperkeratosis nucleus in the center, another gland on the left, h / e staining, magnification × 140 (G); gland colony, h / e staining, magnification × 70 (H); glands containing brown pigment producing cells, possibly bile pigment surrounded by adipose tissue and mesenchymal cells, h / e staining, magnification × 140 (I).

DETAILED DESCRIPTION OF THE INVENTION

Before the present composition, methods and methods of cultivation are described, it should be understood that the present invention is not limited to the specific compositions, methods and experimental conditions described, since these compositions, methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of explaining particular embodiments of the present invention and is not intended to be limiting, since the scope of the present invention is limited only by the appended claims.

Used in this document and the attached claims, the singular formulas “a”, “an” and “the” include the plural, unless the context clearly indicates otherwise. For example, references to a “method” include one or more methods and / or steps of the type described herein, which will be apparent to those skilled in the art after reading the present description and the like.

The present invention describes at least two different approaches for the production of human HLA-homozygous parthenogenetic stem cell lines suitable for transplant based stem cell therapy.

In one embodiment of the present invention, one cell line is obtained from an HLA-homozygous donor, including the use of A23187 and 6-DMAP during oocyte activation, blocking the displacement of the 2nd polar body and, thus, the preservation of the entire genetic material of the MII oocyte. The HLA genotype of stem cells obtained from these oocytes corresponded to the donor genotype (Revazova et al., Cloning Stem Cells (2007) 9 (3): 432-449). In a related aspect, the hpSC-Hhom-1 diploid line from an HLA homozygous donor can also be obtained.

In another embodiment of the present invention, HLA-homozygous embryonic stem cells can be obtained from HLA-heterozygous oocyte donors, including the use of parthenogenetic activation of oocytes in combination with A23187 and puromycin, which allows the displacement of the 2nd polar body. The activated oocyte, therefore, contained only half of the set of chromosomes in metaphase II, which allowed the formation of a homozygous genotype. For example, 80 percent of human oocytes activated by a combination of calcium ionophore and puromycin exhibit pronucleus with the displacement of the second polar body. In addition, cytogenetic analysis shows that 78 percent contain a normal haploid set of chromosomes (Yamano S., et al., J. Med. Investigation (2000) 47 (1-2): 1-8). In addition, human oocytes activated by A23187 and puromycin showed one pronucleus and two polar bodies with a haploid set of chromosomes (Nakagawa K., et al., Zygote (2001) 9: 83-88). The indicated pronuclear parthenot with a normal haploid set of chromosomes was developed on mouse models. (Nakagawa K., et al., Zygote (2000) 8: 203-208).

The use of this protocol results in numerous diploid cell lines from oocytes isolated from HLA-heterozygous donors (for example, without limitation, hpSC-Hhom-2, hpSC-Hhom-3, hpSC-Hhom-4).

Without being bound by theory, HLA genotyping data suggest that the HLA haplotype is inherited exclusively from one of the donor's parents. Analysis of SNP data suggests that these three cell lines are homozygous throughout the genome as estimated by SNP analysis. For example, the examples of cell lines described in the present invention (see Example 1) have a diploid karyotype, consistent with earlier work in which diploid stem cell lines were obtained from haploid mouse embryos (Kaufman et al., J. Embryol. Exp Morphol. (1983) 73: 249-261).

The exact mechanism and time of doubling of the haploid genetic material after activation of the oocyte is unclear, but not limited to theory, it seems that DNA replication is observed in the absence of fragmentation or cell division. Earlier studies suggest that 80% of parthenogenetically activated mouse oocytes retain their haploid state until the morula stage, after which stem cell lines obtained from these embryos become diploid (Kaufman M.H. et al., Above).

In addition to substitution therapy, a repository of cells and tissues derived from hpSC-Hhom lines can be invaluable for treating genetic disorders. According to the Online Mendelian Inheritance in Maine (OMIM), Johns Hopkins University, and the National Center for Biotechnology Information (NCBI), there are more than 100 genetic disorders, and their list continues to grow. Examples include Alzheimer's disease, diabetes, bazedovo disease, hemophilia, Huntington's disease, muscular dystrophy, Parkinson's disease, sickle cell anemia, phenylketonuria (PKU) and severe combined immunodeficiency (SCID). In these situations, it will be important to use cell lines obtained from donors that do not carry the same genetic defect.

"Differentiation" refers to the change that is observed in the cells and leads to the acquisition by the cells of certain specialized functions and to the loss of ability to change to some other specialized functional units. Cells capable of differentiation may be totipotent, pluripotent or multipotent cells. Differentiation may be partial or complete with respect to mature adult cells.

"Gynogenesis" refers to the production of an embryo containing distinguishable trophoblast and internal cell mass, which are the result of activation of a cell, such as an oocyte, or other embryonic cell type, containing mammalian DNA of a completely female nature, preferably human female nature, for example, a primate oocyte DNA, which is or non-human. Said female mammalian DNA may be genetically modified, for example, by insertion, deletion or substitution of at least one DNA sequence, or may be unmodified. For example, DNA can be modified by insertion or deletion of the desired coding sequences or sequences that stimulate or inhibit embryogenesis. Typically, this embryo is obtained by in vitro activation of an oocyte that contains DNA of a completely female nature. Gynogenesis is included in the concept of parthenogenesis, which is defined below. It also includes activation methods in which sperm DNA does not contribute to the activated oocyte DNA.

In a related aspect, oocytes are obtained from superovulation subjects prepared for IVF. “Superovulation” techniques, such as administering hormones to a female subject, used in IVF, are designed to stimulate the ovaries to produce multiple eggs (oocytes), rather than a single egg, as is usually the case with the natural cycle.

Medicines required to increase egg production may include, but are not limited to, the following drugs: Lupron (gonadotropin releasing hormone agonist), Orgalutran, Antagon or Cetrotide (gonadotropin releasing hormone antagonist), Follistim, Bravelle, or Gonal-F ( FSH, follicle-stimulating hormone), Repronex (a combination of FSH and LH, luteinizing hormone) and Pregnyl or Novarel (hCG, human chorionic gonadotropin).

In a related aspect, the selection of eggs can be carried out under transvaginal ultrasound control. To carry out this procedure, a needle is inserted (for example, under iv sedation) through the wall of the vagina into the ovaries using ultrasound to detect the location of each follicle. The follicular fluid is removed and placed in a test tube to obtain eggs.

"Parthenogenesis" ("parthenogenetically activated" and "parthenogenetically activated" are used interchangeably) is a process by which the activation of the oocyte in the absence of sperm penetration is carried out and refers to the development of the embryo at an early stage, including trophoblast and the mass of internal cells, which is obtained by activation of the oocyte or an embryonic cell, for example a blastomere comprising DNA of a completely female nature. In a related aspect, “parthenot” refers to a cell obtained by said activation. In another related aspect, a “blastocyst” refers to the stage of fragmentation of a fertilized or activated oocyte, including a hollow ball of cells consisting of external trophoblast cells and an internal cell mass (ICM). In another related aspect, “blastocyst formation” refers to a process that occurs after fertilization or activation of an oocyte, during which the oocyte is subsequently cultured in the media for the period of time required for its development into a hollow ball of cells consisting of external trophoblast cells and ICM (e.g., 5 to 6 days).

In one embodiment of the present invention, a method for creating a line of cloned human embryonic stem cells by parthenogenetically activated oocytes is described. While parthenogenesis is not a rare form of reproduction in nature, mammals are not able to reproduce in this way. However, 10% of spontaneous parthenogenesis can be detected in the oocytes of the LT / Sv strain of inbred mice (Hoppe and Illmensee, Proc. Natl. Acad. Sci. USA (1982) 79: 1912-1916), and spontaneous parthenogenesis causes the occurrence of cystic drifts in humans ( Berkowitz and Goldstein, New Eng. J. Med. (1996) 335 (23): 1740-1748). In oocytes from placental mammals, in vitro parthenogenesis can be induced; however, the embryo does not develop successfully.

After parthenogenetic activation of the mammalian oocytes and transfer of the activated oocyte to the surrogate mother’s organism, embryo survival is limited: ten days in mice, 21 days in sheep, 29 days in pigs and 11.5 days in rabbits (Kure-bayashi et al., Theriogenology (2000 ) 53: 1105-1119; Hagemann et al., Mol. Reprod. Dev. (1998) 50: 154-162; Ozil and Huneau, Development (2001) 128: 917-928; Surani and Barton, Science (1983) 222 : 1034-1036). The reason for this arrested development is probably genetic imprinting. It has been shown that the maternal and paternal genomes are epigenetically distinct and that both sets are required for the successful development of the embryo (Surani, Cell (1998) 93: 309-312; Sasaki et al., (1992) 6: 1843-1856). If not limited to theory, in parthenotes, all genetic material must be of maternal origin and, therefore, will not have paternal imprinting. Paternal imprinting is thought to be responsible for the development of extraembryonic tissue, such as the development of trophoblast tissue after fertilization of an enucleated oocyte (Surani and Barton (1983), supra). In animals, therefore, enucleated zygotes may be suitable for nuclear transfer followed by parthenogenetic activation.

Parthenotes of mammals undergo only limited development and ultimately the death of the embryo. In Macao fascicular, only 14 percent of stage II metaphase oocytes after in vitro parthenogenetic activation developed to the blastocyst stage after 8 days of cultivation (Vrana et al., Proc. Natl. Acad. Sci. USA (2003) 100 (suppl. 1): 11911 -11916).

Embryos formed in spontaneously activated parthenotes in virgin females of the LT / Sv strain of inbred mice die within a few days. When nuclei are transferred from cells including the internal cell mass (ICM) of these embryos to fertilized enucleated oocytes of C57BL / 6j mice, cloned mice with the LT / Sv genome are obtained (Hoppe and Illmensee, Proc. Natl. Acad. Sci. USA (1982) 79: 1912-1916). Thus, the use of fertilized oocytes involves the termination of parthenot. In one aspect, a fertilized enucleated human oocyte can be used to support the development of a parthenogenetic embryo containing a donor nucleus to the blastocyst stage.

"Pluripotent cell" refers to a cell obtained from an embryo obtained by activation of a cell containing DNA of a completely female or male nature that can be cultured in vitro for a long, theoretically indefinite period of time in an undifferentiated state, which can give rise to various differentiated tissue types, those. ectoderm, mesoderm and endoderm. The pluripotent state of the cells is preferably maintained by culturing the inner cell mass or cells derived from the inner cell mass, an embryo obtained by the androgenetic or gynogenetic method under suitable conditions, for example, culturing fibroblasts on the feeding sublayer or another feeding sublayer or culture, which includes leukemia inhibiting factor (LIF) . The pluripotent state of these cultured cells can be confirmed by various methods, for example (i) confirmation of the expression of markers characteristic of pluripotent cells; (ii) the production of hybrid animals that contain cells expressing the genotype of pluripotent cells; (iii) injection of cells into animals, such as SCID mice, with the production of various differentiated cell types in vivo; and (iv) observing cell differentiation (for example, when cultured in the absence of a feeding sublayer or LIF) into embryoid bodies and other differentiated cell types in vitro.

“Diploid cell” refers to a cell, for example an oocyte or blastomere, having a diploid DNA set of a completely male or female nature.

“Haploid cell” refers to a cell, for example an oocyte or blastomere, having a haploid DNA set of a completely male or female nature.

“Activation” refers to a process in which a fertilized or unfertilized oocyte, for example, without limitation, located in the metaphase II of meiosis, undergoes a process that usually involves the separation of chromatid pairs, the expulsion of the second polar body, resulting in an oocyte having a haploid number of chromosomes each with one chromatid. Activation includes methods whereby a cell containing DNA of a completely male or female nature is induced to develop into an embryo, which has a distinct internal cell mass and trophoblast, which is suitable for the production of pluripotent cells, but which itself is probably not capable of developing into a viable offspring. Activation can be carried out, for example, under one of the following conditions: (i) under conditions that do not cause the ejection of the second polar body; (ii) under conditions that cause the ejection of the second polar body, but the ejection of the second polar body is inhibited; or (iii) under conditions that inhibit the first cell division of the haploid oocyte.

"Metaphase II" refers to the stage of cell development, during which the DNA of the cells consists of a haploid set of chromosomes, each of which is represented by two chromatids. For the present invention, the suppression of the second meiotic division after parthenogenetic activation of human oocytes in metaphase II and the generation of a diploid embryo results in the generation of MHC heterozygous phESCs.

Typically, the oxygen tension in the fallopian tube and uterine uterus of mammals is much lower than half the oxygen tension in a normal atmosphere (Fisher and Bavister, J. Reprod. Fertil. (1993) 99: 673-679; Kaufman et al., Comp. Biochem. Physiol. Comp. Physiol. (1994) 107: 673-678). For successful cultivation of human embryos after IVF, oxygen concentrations of 20% as well as 5% were used. However, an increase in oxygen can generate reactive oxygen species that apoptosis can induce (Van Soom et al., Theriogeology (2002) 57: 1453-1465). A low oxygen concentration has been reported to increase the viability of embryos prior to implantation, contribute to their normal development, and more often ensure the formation of healthy blastocysts, as evidenced by the greater number of cells and well-formed internal cell mass (ICM) (Dumoulin et al., Hum. Reprod. (1999) ) 14: 465-469). In earlier studies, parthenogenetic human embryos developed in vitro using gas mixtures with a high (20%) oxygen content (Lin et al., Stem Cells (2003) 21: 152-161; Cibelli et al., J. Reg. Med. (2001) 2: 25-31).

In one embodiment of the present invention, methods for isolating ICM and culturing phESC included the use of human skin fibroblasts, and cells were expanded using human umbilical cord blood serum (HUCBS) instead of animal blood serum and used as feeding cells. Derivation and cultivation of phESC lines can be carried out in VitroHES medium (Vitrolife), developed for hESC cultivation, with the addition of HUCBS. For example, using HUCBS to produce hESC has a positive effect on ICM proliferation and phESC proliferation (for example, phESC growth in VitroHES was better with HUCBS than without it). In addition, the isolation of ICM from whole blastocysts by mechanical sections from the outgrowth of trophoblast seems to be a more gentle and preferred method compared to immunosurgery and trypsin treatment. In addition, this method allowed to exclude interaction with reagents of animal origin.

Although the phESC lines of the present invention possess typical characteristics of hESC lines, they also exhibit unique properties, including genotypes that are almost identical to the genotypes of oocyte donors, as can be seen from the parthenogenetic line of monkey ES cells of Cyno-1 (Vrana et al. 2003, Proc. Natl. Acad. Sci. USA (2003) 100: 11911-11916). As such, constructing hESC lines from parthenogenetic embryos can be an excellent way to generate MHC-compatible and possibly histocompatible embryonic stem cells compared to SCNT.

As previous studies on murine and monkey parthenogenetic stem cells have shown, these cells can form teratomas with derivatives from all three embryonic germ layers (Lin et al., 2003, supra; Vrana et al., Supra). Monkey parthenogenetic embryonic stem cells under different cultivation conditions differentiated into nerve cells and functional dopaminergic and serotonergic neurons (Vrana et al., Supra). The phESCs of the present invention can also differentiate into derivatives of all three germ layers in vitro and in vivo and are pluripotent. In addition, phESC embryoid bodies were able to give rise to contracting cells similar to cardiomyocytes.

The present invention demonstrates a method for creating parthenogenetic human embryonic stem cells, experimental data on these cells show that phESCs can differentiate into functional cells, which can be of great importance for the treatment of degenerative diseases in humans.

In one embodiment of the present invention, oocytes in metaphase II are activated by incubating the oocytes at various O ₂ pressures in the environment. In a related aspect, the low voltage of O ₂ gas is created by a gas mixture comprising O ₂ at a concentration of about 2%, 3%, 4% or 5%. In another related aspect, the gas mixture comprises about 5% CO ₂ . In addition, the gas mixture includes approximately 90% N ₂ , 91% N ₂ or 93% N ₂ . The specified gas mixture should be distinguished from air with 5% CO ₂ , which contains approximately 5% CO ₂ , 20% O ₂ and 75% N ₂ .

"O ₂ partial pressure" refers to the partial pressure (pressure that one component of the gas mixture creates) of oxygen in a liquid (i.e., in a liquid or gas). Low pressure exists when the partial pressure of oxygen (pO ₂ ) is low, and high pressure exists when pO ₂ is high.

"Certain environmental conditions" refers to the environment surrounding cultured cells, in which the concentrations of the components required for optimal growth are described in detail. For example, depending on the use of cells (for therapeutic purposes, for example), removing cells from conditions that contain foreign proteins is important; those. culture conditions are conditions that do not contain components of animal origin or do not contain proteins of animals that are not human. In a related aspect, “in vitro fertilization media (IVF)” refers to a nutrient system that contains chemically defined substances or in which fertilized oocytes can be grown.

“Extracellular matrix (ECM) substrates” refer to the surface under the cells that supports optimal growth. For example, these ECM substrates include, but are not limited to, Matrigel, laminin, gelatin, and fibronectin substrates. In a related aspect, these substrates may include collagen IV, entactin, proteoglycan heparin sulfate, various growth factors (e.g., bFGF, epidermal growth factor, insulin-like growth factor-1, platelet-derived growth factor, nerve growth factor and TGF-β-1).

"Embryo" refers to an embryo that is formed as a result of activation of a cell, such as an oocyte or other embryonic cell containing DNA of a completely male or female nature, which optionally can be modified, which includes a distinguishable trophoblast and an internal cell mass that cannot produce viable offspring and in which the DNA is completely male or female in nature. The internal cell mass or cells that it contains are suitable for the production of pluripotent cells, as previously defined.

“Internal cell mass (ICM)” refers to the inside of an embryo that gives rise to fetal tissues. In the present invention, these cells are used to create a permanent source of pluripotent cells in vitro. In addition, ICM includes the interior of the embryo, which occurs as a result of androgenesis or gynogenesis, i.e. embryos that result from the activation of cells containing DNA of a completely male or female nature. Said DNA may be, for example, human DNA, for example, human oocyte or human sperm DNA, which may or may not be genetically modified.

"Trophoblast" refers to another part of an embryo that is in an early stage of development, which gives rise to placental tissues, including embryonic tissue, which occurs as a result of androgenesis or gynogenesis, i.e. embryos that result from the activation of cells containing DNA of a completely male or female nature, for example, an oocyte or human sperm.

"Differentiated cell" refers to a non-embryonic cell that is in a specific differentiated, i.e. non-embryonic condition. The three earliest differentiated cell types are endoderm, mesoderm, and ecdoderm.

“Almost identical” refers to the quality of similarity regarding a particular characteristic that is so close that it is pretty much the same within its ability to measure differences (for example, through HLA typing, SNP analysis, etc.).

“Histocompatible” refers to the extent to which an organism will tolerate a transplant of foreign tissue.

“Genomic imprinting” refers to a mechanism by which a number of genes in a genome are expressed mono-allelic, according to its origin from the parent.

“Homoplasmy,” including grammatical variants, refers to the presence of the same type of mitochondrial DNA (mtDNA) in a cell or individual.

“Heteroplasmia,” including grammatical variants, refers to the presence of a mixture of more than one type of mitochondrial DNA (mtDNA) in a cell or individual.

"Parthenogenetic" refers to one or more cells or individuals from which another cell or individual originates and in relation to which it remains secondary.

"Mechanical isolation" refers to the process of separation of cellular aggregates by physical forces. For example, this process eliminates the use of enzymes (or other degrading cell products) that may contain materials not derived from humans.

In the natural environment, immature oocytes (ovules) from the ovary undergo a maturation process, as a result of which development through meiosis to metaphase II of meiosis occurs. Oocytes then stop in metaphase II. In metaphase II, the DNA in the cell contains a haploid number of chromosomes, each of which is represented by two chromatids.

These oocytes can be stored using indefinitely using cryopreservation, for example, without limitation, using microinjection of sugar.

In one embodiment of the present invention, a method is described for producing human stem cells from a cryopreserved oocyte or parthenot, comprising microinjecting a cryopreservative agent into the cytoplasm of an oocyte or parthenot, freezing the oocyte or parthenot to a cryogenic temperature to put it into a dormant state, storing the oocyte or parthenot in a dormant state, thawing the oocyte or parthenotes, parthenogenetic activation of oocytes with a high partial pressure of O ₂ in the presence of the ionophore with subsequent contacting the oocyte with an inhibitor of serine threonine kinase with a low partial pressure of O _2, culturing the activated oocyte or parthenotes to formation of the blastocyst, isolation of the inner cell mass (ICM) of the blastocyst and culturing the ICM cells on a layer of human feeder cells, where culturing the ICM cells is carried out at high partial pressure of O ₂ .

In one aspect, oocytes obtained by the method described above are transferred to a modified isotonic IVF medium coated with embryo-tested mineral oil (Sigma), or any other suitable medium. If desired, oocytes can be incubated with extracellular sugar at the same concentration as planned for microinjection. For example, to inject 0.1 M sugar, oocytes can be balanced in DMEM / F-12 with 0.1 M sugar. In one aspect, the cryopreservation agent comprises a lower concentration of Na ^+, than standard DMEM (i.e., medium with low content of Na ^+). In a related aspect, the cryopreservation agent comprises a higher concentration of K ⁺ than standard DMEM (i.e., with a high content of K ⁺ ). In yet another related aspect, the cryopreservation agent comprises a lower Na ⁺ concentration and a higher K ⁺ concentration than standard DMEM (i.e., media with a low Na ⁺ content and a high K ⁺ content). In one aspect, the cryopreservation agent comprises an organic buffer, including, without limitation, HEPES. In another aspect, the cryopreservation agent includes components that inhibit an apoptotic protein (e.g., capase).

Alternatively, oocytes can optionally be balanced using any other substantially non-permeable, soluble agent, such as NaCl, to reduce their cell volume prior to microinjection. The indicated decrease in cell volume can lead to a smaller final volume of oocytes that received microinjection compared to oocytes that were not incubated in hypertonic media prior to microinjection. The indicated smaller volume can minimize any potential adverse effect of oocyte swelling. The specified general procedure for preparing cells for microinjection can also be used for other types of cells (for example, activated oocytes, hES cells, etc.).

Then microinjection into the oocytes of the cryopreservation agent. Equipment and procedures for microinjection are well described, and equipment for microinjection, known for use for injection of small molecules into cells, can be used in the present invention. As an example of a microinjection step, microinjection into oocytes can be carried out at a pressure of 10 inches per square meter. inch for 30 milliseconds. Another example of a standard microinjection technique is the method described by Nakayama and Yanagimachi (Nature Biotech. 16: 639-642, 1998).

A cryopreservation agent suitable for this method includes any chemical agent that has cryoprotective properties and is usually non-permeable. In particular, the cryopreservation agent may include sugars both individually and in admixture with other conventional cryopreservation agents. Carbohydrate sugars, such as trehalose, sucrose, fructose and raffinose, can be microinjected to concentrations less than or equal to about 1.0 M, more preferably less than or equal to about 0.4 M. In one aspect, the concentration is from 0.05 to 0, 20 M inclusive. In addition, extracellular sugar or a traditional cryopreservation agent can be added before storage. If the cells were incubated in a hypertonic solution before microinjection, the non-penetrating agent can be allowed to remain in the media after microinjection, or it can be removed from the media by washing the cells with media containing a lower concentration of the indicated solute or not containing it at all.

Some sugars or polysaccharides, which usually do not penetrate cell membranes because they are too large to pass through the membrane, have excellent physicochemical and biological properties for cryopreservation. While these sugars generally do not permeate through cell membranes by themselves, using the method described herein, these generally non-permeable sugars can be microinjected intracellularly to produce a beneficial effect.

Non-permeable sugars having a stabilizing or preserving effect on cells that are particularly suitable as a cryopreservation agent in the present method include sucrose, trehalose, fructose, dextran and raffinose. Among these sugars, trehalose, a non-reducing glucose disaccharide, has been shown to be extremely effective in stabilizing cell structures at low concentrations. The addition of extracellular glycolipids or glycoproteins can also stabilize the cell membrane.

After microinjection of the cryopreservation agent, the cells are prepared for storage. A number of freezing and / or drying methods can be used to prepare cells for storage. In particular, three approaches are described in this document: drying under vacuum or air, lyophilization, and freeze / thaw protocols. Drying processes have the advantage that stabilized biological material can be transported and stored at room temperature.

Typically, oocytes loaded using DMSO from 1 to 2 M are cooled at a very low cooling rate (from 0.3 to 0.5 ° C per minute) to an intermediate temperature (from -60 ° C to -80 ° C) before immersion in liquid nitrogen for storage. The sample can then be stored at the indicated temperature.

Suspended material can be stored at cryopreservation temperatures, for example, leaving the bottles in liquid nitrogen (LN ₂ ) for a desired period of time.

Protocols for drying in vacuum or air and for lyophilization of proteins are well described (Franks et al., “Materials Science and the Production of Shelf-Stable Biologicals”, BioPharm, October 1991, p. 39; Shalaev et al., “Changes in the Physical State of Model Mixtures during Freezing and Drying: Impact on Product Quality, ”Cryobiol. 33, 14-26 (1996)), and these protocols can be used to prepare cell suspensions for storage by the method described herein. In addition to air drying, other convective drying methods that can be used to remove water from cell suspensions include a convective flow of nitrogen or other gases.

An example evaporative vacuum drying protocol suitable for the method of the present invention may include placing 20 μl of material in each of the cells of 12-cell plates and vacuum drying for 2 hours at room temperature. Of course, other drying methods can be used, including drying the cells in vials. Cells prepared by this method can be stored dry and rehydrated by dilution in DMEM or any other suitable medium.

The method of the present invention using freeze drying to prepare cells for storage begins with freezing the cell suspension. While freezing methods known in the art can be used, the simple immersion freezing method described herein for the freeze-thaw method can also be used for the freezing step in the lyophilization protocol.

After freezing, a two-stage drying process can be used. In the first step, sublimation energy is added to evaporate the frozen water. Secondary drying is carried out after sublimation from a sample of pure crystalline ice. Freeze dried cells can be stored and hydrated as described above for vacuum drying. Viable cells can then be restored.

After restoration of the cells from a frozen or dried state, any external cryopreservation agent may optionally be removed from the culture media. For example, media can be diluted by adding appropriate media with a lower concentration of cryopreservation agent. For example, reconstituted cells can be incubated for approximately five minutes in media containing a lower concentration of sugar than in those media used to store cells. For this incubation, the media may contain the same sugar that was used as the cryopreservation agent; another cryopreservation agent, such as galactose, or any other practically non-permeable dissolving agent. To minimize any osmotic shock caused by a decrease in osmolarity of the media, the concentration of the extracellular cryopreservation agent can be slowly reduced by performing the indicated dilution step many times, each time with a lower concentration of cryopreservation agent. The indicated dilution steps can be repeated until an extracellular cryopreservation agent remains, or until the concentration of cryopreservation agent or osmolality of the media decreases to the desired level.

Parthenogenetically activated oocytes, blastocysts, ICM, autologous stem cells and differentiated cells obtained from them can be stored or “create a cell bank” in a way that allows cells to be revived when necessary in the future. An aliquot of parthenogenetically activated oocytes and autologous or allogeneic stem cells can be extracted at any time to grow many undifferentiated cells in cultures, and then differentiate into a specific cell type or tissue type, and then they can be used to treat a disease or to replace poorly functioning tissues in a subject . In one aspect, the cells are obtained by the parthenogenetic route from a donor, the cells can be stored in such a way that the individual or his close relative can access the cells for an extended period of time. In another aspect, the cells are obtained by the parthenogenetic route from a donor that is homozygous for the HLA haplotype, which is common in the human population; the cells can be stored in such a way that an individual with the same or similar HLA haplotype can have access to the cells for an extended period of time. In one aspect, the cells are obtained by the parthenogenetic route from a donor that has an HLA haplotype that is common in the human population; the cells can be stored in such a way that an individual with the same or similar HLA haplotype can have access to the cells for an extended period of time.

In one embodiment of the present invention, a cell bank is created for storing oocytes, blastocysts, ICM and / or samples of autologous or allogeneic stem cells and their differentiated derivatives obtained by the parthenogenetic method. In another embodiment of the present invention, methods of using said cell bank are described. US Patent Application Publication No. 20030215942, which is incorporated herein by reference in its entirety, describes an example of a stem cell bank system.

The use of methods such as the methods described above, isolation and in vitro propagation of parthenogenetically activated oocytes, blastocysts, ICM and samples of autologous or allogeneic stem cells and their differentiated derivatives, as well as their cryopreservation facilitate the creation of a “bank” suitable for transplantation of human stem cells. Since it is possible to store smaller aliquots of cells, the bank storage procedure can take up relatively small space. Thus, cells from many individuals can be stored or “banked" on a short or long term basis at a relatively low cost.

In one embodiment of the present invention, part of the sample is made available for testing, both before and after processing and storage.

The present invention also relates to methods for labeling or indexing parthenogenetically activated oocytes, blastocysts, ICM and / or samples of autologous or allogeneic stem cells and their differentiated derivatives in such a way that when it is necessary to determine the location of the sample, it can be easily removed. Any indexing and retrieval system can be used to achieve this goal. Any suitable type of storage system can be used in such a way that parthenogenetically activated oocytes, blastocysts, ICM and / or autologous or allogeneic stem cell samples and their differentiated derivatives can be stored. Systems can be designed to store individual samples, or can be designed to store hundreds, thousands, and even millions of samples of different cells.

Stored parthenogenetically activated oocytes, blastocysts, ICM and / or autologous or allogeneic stem cell samples and their differentiated derivatives can be indexed for reliable and accurate removal. For example, each sample can be marked with an alphanumeric code, barcode, or any other method, or a combination thereof. There may also be an accessible and readable list of information that makes it possible to identify each parthenogenetically activated oocyte, blastocyst, ICM and / or sample of autologous or allogeneic stem cells and their differentiated derivatives and determine their location in the bank and makes it possible to identify the source and / or type a sample of cells that is outside the bank. The specified indexing system can be arranged in any known manner, for example, manual or non-manual, for example, you can use ordinary computer programs.

In one embodiment of the present invention, cell samples are organized using an indexing system so that cell samples are available for use by the donor whenever they are needed. In other embodiments of the present invention, cell samples can be used by individuals who are relatives of the original donor. In alternative embodiments of the present invention, cell samples can be used by individuals having HLA haplotypes that match the HLA haplotypes of cell samples. Once entered into the indexing system, a cell sample can be made available for compatibility search purposes, for example, for a compatibility program, it will identify an individual with the same type of information and the individual will have the choice of a compatible sample.

A bank storage system may include a system for storing a plurality of records related to a plurality of individuals and a plurality of cell samples. Each entry may contain type information, genetic information, or phenotypic information associated with cell samples or specific individuals. In one embodiment of the present invention, the system will include a cross-compatibility table that combines the types of samples with the types of individuals who wish to obtain a sample.

In one embodiment of the present invention, information is stored in a database system for each parthenogenetically activated oocyte, blastocyst, ICM and / or sample of autologous or allogeneic stem cells or their differentiated derivatives in a bank. Certain information is stored in association with each sample. Information may be associated with a specific donor, for example, donor identification and donor medical history. For example, each sample may be HLA typed, and HLA type information may be stored in association with each sample. Stored information may also be available information. The information stored with each sample is searchable and identifies the sample in such a way that it can be located and immediately provided to the customer.

Accordingly, embodiments of the present invention use computer database systems that contain information such as a donor, date of registration, type of cells provided, types of cell surface markers presented, HLA cell types, genetic information related to the donor, or other relevant information as well as storage details such as storage records and the location of stored samples and other useful information.

The term “computer-based system” refers to computer hardware, software, and any database that is used to store, search, and retrieve information about stored cells. A computer-based system preferably includes storage means described above and a processor for accessing and manipulating data. The hardware of the computer-based systems of this embodiment of the present invention includes a central processing unit (CPU) and a database. An experienced person can easily appreciate that any of the currently available computer-based systems is suitable.

In one embodiment of the present invention, the computer system includes a processor coupled to a bus that is connected to main memory (preferably RAM) and a number of different secondary storage devices, such as a hard disk and removable storage media. A removable storage medium may be, for example, a floppy drive, a DVD drive, an optical drive, a compact disk drive, a magnetic tape drive, and the like. A removable storage medium such as a floppy disk, a compact disk, a magnetic tape or the like containing control logic and / or data recorded thereon can be inserted into a removable storage device. The computer system includes a suitable program for reading control logic and / or data from a removable storage medium once inserted into a storage removable device. Information regarding a parthenogenetically activated oocyte, blastocyst, ICM and / or sample of autologous or allogeneic stem cells can be stored in a well-known manner in the main memory, any of the secondary storage devices and / or removable storage media. A program for accessing and processing specified data (such as a search tool, a comparison tool, etc.) is constantly located in the main memory at runtime.

As used herein, a “database” refers to a memory that can store any useful information relating to collections of parthenogenetically activated oocytes and / or collections of autologous or allogeneic stem cells, including their differentiated derivatives, and to donors.

Data related to stored parthenogenetically activated oocyte, blastocyst, ICM and / or autologous or allogeneic stem cells and their differentiated derivatives can be stored and used in a number of data processor programs in a number of formats. For example, data can be stored as text in a text program such as Microsoft WORD or WORDPERFECT, ASCII file, html file or pdf file, in a number of database programs known to those skilled in the art, such as DB2, SYBASE or ORACLE.

A “search program” refers to one or more programs that are executed on a computer-based system to search for parts or compare information related to cryopreserved samples within a database. An “issuing program” refers to one or more programs that may be executed in a computer-based system to identify parameters of interest in a database. For example, a dispensing program can be used to find samples that match a particular profile, samples that have specific markers or DNA sequences, or to find the location of samples that match specific individuals.

There is no upper bound on the number of cell samples that can be stored in one cell bank. In one embodiment of the present invention, hundreds of products from various individuals will be stored in a single bank or warehouse. In another embodiment of the present invention, up to millions of products can be stored in one storage facility. One storage facility can be used to store samples of parthenogenetically activated oocytes and / or autologous or allogeneic stem cells, or multiple storage facilities can be used.

In some embodiments of the present invention, the storage facility may have means for implementing any method of organizing and indexing stored cell samples, such as, for example, automated robotic delivery mechanisms and mechanisms for manipulating cell samples. The construction may include micromanipulation devices for processing cell samples. Known conventional techniques can be used to efficiently store and deliver cell samples. Examples of technologies include, but are not limited to, Machine Vision, Robotics, Automated Guided Vehicle System, Automated Storage, and Retrieval Systems, Computer Integrated Manufacturing, Computer Aided Process Planning, Statistical Process Control, and the like.

The type of information or other information associated with an individual who needs a sample can be entered into a system that can be used to identify a suitable product, such as, for example, a database system, an indexing system, and the like. After entering into the system, it is possible to determine the correspondence between the type of individual and the donor cell sample. In preferred embodiments of the present invention, the donor sample is derived from the same individual who needs the sample. However, donor / recipient matches that are similar but not identical can also be used. A compatible sample is available to an individual having a compatible type identifier. In one embodiment of the present invention, the identifying information of an individual is stored in association with a sample of cells. In some embodiments of the present invention, a matching process is observed at a time approximately corresponding to the sampling time, or may be observed at any time during processing, storage, or the need for a sample. Accordingly, in some embodiments of the present invention, a matching process is observed before the individual actually needs a cell sample.

When a parthenogenetically activated oocyte, blastocyst, ICM and / or samples of autologous or allogeneic stem cells, including their differentiated derivatives, are required by an individual, they can be delivered and made available for research, transplantation or other purposes within minutes if necessary. The sample can also be subsequently processed to prepare it for transplantation or for other needs.

Typically, an oocyte that stops in metaphase II ovulates and is fertilized by sperm. Sperm initiates the completion of meiosis in a process called activation. During activation, the chromatid pairs are separated, the secondary polar body is pushed out, and the oocyte retains the haploid number of chromosomes, each with one chromatid. Sperm introduces another haploid set of chromosomes with the formation of a fully diploid cell with single chromatids. Chromosomes then progress through DNA synthesis during the first cell cycle. These cells then develop into embryos.

In contrast, the embryos described herein develop as a result of the artificial activation of cells, usually mammalian oocytes or blastomeres, containing DNA of a completely male or female origin. As discussed previously, in the section "prior art", in the literature there are reports of numerous methods of artificial activation of unfertilized oocytes. These methods include physical methods, for example mechanical methods, such as injection, manipulation or oocytes in culture, thermal methods, such as cooling and heating, repeated electrical impulses, enzymatic effects, such as trypsin, pronase, hyaluronidase, osmotic effects, ionic effects such as exposure to divalent cations and calcium ionophores such as ionomycin and A23187, the use of anesthetics such as ether, ethanol, tetracaine, lignocaine, procaine, phenothia zine, tranquilizers such as thioridazine, trifluoroorazine, flufenazine, chlorpromazine, the use of protein synthesis inhibitors such as cycloheximide, puromycin, the use of phosphorylation inhibitors such as protein kinase inhibitors such as staurosporine, 2-aminopurine, sphingosine, and also DMAP, other methods.

These activation methods are well known in the art and are discussed, for example, in US Pat. No. 5,945,577.

In one embodiment of the present invention, a human cell in metaphase II, typically an oocyte or blastomere including DNA of a completely male or female origin, is artificially activated to artificially activate the oocytes.

In a related aspect, an activated cell, such as an oocyte that is diploid, is allowed to develop into an embryo that includes trophoblast and internal cell mass. This can be done using known methods and culture media that facilitate the development of blastocysts.

After the gynogenetic embryos were cultured to form a distinguishable trophoblast and inner cell mass, the cells of the inner cell mass are then used to obtain the desired pluripotent cell lines. This can be accomplished by transferring cells obtained from the inner cell mass, or the whole cell mass into a culture that inhibits differentiation. This can be accomplished by transferring the internal cell mass to a supply layer that inhibits differentiation, for example, fibroblasts or epithelial cells, such as fibroblasts derived from postnatal human tissues and the like, or other cells that produce LIF. Other factors / components can be used to create suitable culture conditions to maintain the cells in an undifferentiated state, including, but not limited to, the addition of conditioned media (Amit et al., Developmental Biol. (2003) 227: 271-278), bFGF and TGF- β1 (with or without LIF) (Amit et al., Biol. Reprod. (2004) 70: 837-845), factors that activate the gp130 / STAT3 pathway (Hoffman and Carpenter, Nature Biotech. (2005) 23 (6 ): 699-708), factors that activate the PI3K / Akt pathway, PKB (Kim et al., FEBS Lett. (2005) 579: 534-540), factors that are members of the bone marrow morphogenetic protein (BMP) superfamily (Hoffman and carpen ter (2005), supra), and factors that activate the canonical / β-catenin Wnt signaling pathway (e.g., GSK-specific inhibitor; Sato et al., Nat. Med. (2004) 10: 55-63). In a related aspect, these factors may include culture conditions that include feeder cells and / or ECM substrates (Hoffman and Carpenter (2005), supra).

In one aspect, cells of the inner cell mass are cultured on human postnatal foreskin cells or skin fibroblasts or on other cells that produce a leukemia inhibitory factor, or in the presence of a leukemic inhibitory factor. In a related aspect, the nourishing cells inactivate before seeding ICM. For example, nourishing cells can be mitotically inactivated using an antibiotic. In a related aspect, the antibiotic may be, without limitation, mitomycin C. In a related aspect, nourishing cells can be inactivated using radiation.

Cultivation will be carried out under conditions that support cells in an undifferentiated, pluripotent state, over extended periods of time, theoretically infinitely. In one embodiment of the present invention, oocytes are parthenogenetically activated using calcium ionophores at a high O ₂ partial pressure, followed by contact of the oocytes with a serine threonine kinase inhibitor at a low O ₂ partial pressure. The ICM obtained from parthenogenetically activated oocytes is cultured at a high partial pressure of O ₂ when the cells, for example, are maintained using a gas mixture containing 20% O ₂ . In one aspect, “cultivated” refers to capable or suitable for cultivation. In a related aspect, the isolation of ICM is carried out mechanically, after four days of blastocyst culture, when cultured on feeder cells. The specified cultivation, for example, eliminates the need to use materials obtained from sources of animal origin, as is the case in the case of immunosurgery.

In a related aspect, non-animal sera are added to ICM culture media, including, but not limited to, human umbilical cord serum when serum is present in certain media (e.g., IVF from MediCult A / S, Denmark; Vitrolife, Sweden, or Zander IVF, Inc ., Vero Beach, Fl). In another aspect of the environment and the processes in the form in which they are provided, do not contain products of animal origin. In a related aspect, animal products are products, including serum, interferons, chemokines, cytokines, hormones, and growth factors, which are obtained from non-human sources.

The pluripotent state of cells obtained using the present invention can be confirmed in various ways. For example, cells can be tested for the presence or absence of characteristic markers of ES cells. In the case of human ES cells, examples of these markers are identified above and include SSEA-4, SSEA-3, TRA-1-60, TRA-1-81 and OCT4 and are known in the art.

Also, pluripotency can be confirmed by injecting cells into a suitable animal, such as a SCID mouse, and observing the production of differentiated cells and tissues. Another way to confirm pluripotency is to use the subject's pluripotent cells to generate chimeric animals and to observe the contribution of the introduced cells to various cell types. Methods for producing chimeric animals are well known in the art and are described in US Patent No. 6642433.

Another way to confirm pluripotency is to observe the differentiation of ES cells in embryoid bodies and other differentiated cell types when cultured under conditions that promote differentiation (for example, removal of the feeding layers of fibroblasts). This method was used, and it was confirmed that the pluripotent cells of the subject gave rise to embryoid bodies and various differentiated cell types in tissue culture.

The resulting pluripotent cells and cell lines, preferably human pluripotent cells and cell lines that are derived from DNA of a completely female origin, are used for a variety of therapeutic and diagnostic purposes. These pluripotent cells can be used to treat cell transplantation or for gene therapy (subject to genetic modification) to treat numerous disease states.

In this regard, it is known that murine embryonic stem (ES) cells are able to differentiate into almost any cell type. Therefore, human pluripotent (ES) cells obtained in accordance with the present invention have a similar ability to differentiate. The pluripotent cells of the present invention will be induced to differentiate in order to obtain the desired cell types, in accordance with known methods. For example, human ES cells obtained in accordance with the present invention can be induced to differentiate to produce hematopoietic stem cells, muscle cells, heart muscle cells, liver cells, pancreatic islet cells, retinal cells, cartilage cells, epithelial cells, urinary tract cells etc. by culturing these cells in a medium for differentiation and under conditions that ensure the differentiation of cells. Media and methods that allow differentiation of ES cells are known in the art as suitable culture conditions.

For example, Palacios et al., Proc. Natl. Acad. Sci. USA, 92: 7530-7537 (1995) describe the preparation of hematopoietic stem cells from an embryonic cell line using an induction procedure for stem cells, including the initial cultivation of aggregates of these cells in a suspension culture medium containing no retinoic acid, followed by cultivation in the same retinoic acid containing medium, followed by transfer of cell aggregates to a substrate, which involves the attachment of cells.

In addition, Pedersen, J. Reprod. Fertil. Dev., 6: 543-552 (1994) presents a review article that cites numerous articles describing in vitro methods for differentiating embryonic stem cells to produce various differentiated cell types, including, but not limited to, hematopoietic cells, muscle cells, heart muscle cells , nerve cells.

In addition, Bain et al., Dev. Biol., 168: 342-357 (1995) describe in vitro differentiation of embryonic stem cells to produce nerve cells that possess neuronal properties. These references are examples of literature methods for producing differentiated cells from embryonic or stem cells. So, using known methods and culture medium, a specialist can cultivate ES cells that are the subject of this discussion, including those obtained by genetic engineering or transgenic ES cells, to obtain the desired differentiated cell types, for example, nerve cells, muscle cells, hematopoietic cells, etc. P. Pluripotent cells obtained by the methods described herein can be used to produce any desired differentiated cell type.

For example, major histocompatibility complex (MHC) homozygosity of the hpSC-Hhom line may be uniquely suited for therapeutic use. With proper selection of oocyte donors according to their HLA haplotype and HLA haplotype of the biological parents of the donor and using FDA approved protocols, it is possible to create a bank of cell lines whose tissue derivatives collectively could be MHC-compatible with a significant number of individuals. It has been suggested that a panel of only ten HLA-homozygous human embryonic stem cell lines selected by common types can provide full HLA-A, HLA-B and HLA-DR compatibility for 37.7% of recipients in the United Kingdom and favorable compatibility for 67.4% (Taylor CJ et al., Lancet (2005) 366 (9502): 2019-2025). Using calculations for the US population suggests that there are about 200 common haplotypes per racial group (Mori M. Et al., Transplantation (1997) 64: 1017-1027). The hpSC-Hhom-4 line carries the most common haplotype, potentially providing MHC compatibility for almost 5% of individuals in the population. Thus, the hpSC-Hhom line is ideal for creating a repository of differentiated cells and tissues that are HLA-compatible for a population that could be available for immediate clinical use. There may be a concern regarding hematopoietic derivatives that could potentially contribute to graft versus host disease (Billingham, R.E., Harv. Led. (1966) 62: 21-78) in a heterozygous recipient; in this case, patient-specific parthenogenetic stem cells can solve the problem.

The therapeutic uses of differentiated human cells are unparalleled. For example, human hematopoietic stem cells can be used for medical treatment requiring bone marrow transplantation. These procedures are used to treat many diseases, such as advanced malignant tumors, such as ovarian cancer and leukemia, as well as diseases that compromise the immune system, such as AIDS. Hematopoietic stem cells can be obtained, for example, by introducing male or female DNA obtained from a male or female patient having a malignant tumor or AIDS into an enucleated oocyte, obtaining pluripotent cells as described above, and culturing these cells under conditions that favor differentiation , up to receiving hematopoietic stem cells. These hematopoietic cells can be used to treat diseases, including cancer and AIDS.

Alternatively, the pluripotent cells of a subject can be used to treat a patient suffering from a neurological disorder by culturing said cells under differentiation conditions that produce nerve cell lines. Specific diseases that can be treated by transplantation of these human nerve cells include, but are not limited to, for example, Parkinson's disease, Alzheimer's disease, ALS and cerebral palsy. In a specific case of Parkinson's disease, it has been shown that transplanted fetal nerve cells in the brain form the proper compounds with surrounding cells and produce dopamine. This may result in long-term reverse development of symptoms of Parkinson's disease. In a related aspect, nerve cell precursors can be used to resuscitate severely damaged nerve fibers to restore movement after damage to the arms, legs, and spinal cord.

One objective of the present invention is to provide a virtually unlimited source of pluripotent human cells that can be used to produce differentiated cells suitable for autologous transplantation of an oocyte donor or allogeneic transplantation of HLA-compatible recipients. Human embryonic stem cells and their differentiated descendants derived from blastocysts remaining after infertility treatment, or created using NT, are likely to be rejected by the recipient's immune system if they were used for therapy with allogeneic cell transplantation. The stem cells obtained by the parthenogenetic method will produce differentiated cells that can alleviate the significant problem associated with currently existing transplantation methods, i.e. rejection of the transplanted tissue, which can be observed as a result of the host versus transplant or graft versus host reaction with respect to the oocyte donor. Typically, rejection is prevented or reduced by the administration of anti-rejection drugs such as cyclosporine. However, these drugs have significant adverse side effects, such as immunosuppression, carcinogenic properties, as well as a very high cost. Cells obtained by the methods described herein will need to eliminate in some cases and at least reduce in other cases the need for anti-rejection medications relative to the oocyte donor.

Another objective of the present invention is the creation of an almost unlimited source of pluripotent human cells that can be used to obtain differentiated cells suitable for allogeneic transplantation to family members of the oocyte donor (for example, brothers or sisters). Cells will be immunologically and genetically similar to cells of direct members of the oocyte donor family and are thus less likely to be rejected by members of the donor family.

Another objective of this method is that parthenogenetic activation of mammalian oocytes is a relatively simple procedure compared to SCNT and leads to the creation of stem cells with fewer cell manipulations.

Parthenogenetic activation of mammalian oocytes has been shown to be more effective for creating stem cells than methods requiring mechanical manipulation of oocytes (e.g., SCNT).

A disadvantage of SCNT is that subjects with a deficit in mitochondrial respiratory chain activity present phenotypes with striking similarities with the types of pathology commonly found in fetuses and offspring after SCNT (Hiendleder et al., Repro. Fertil. Dev. (2005) 17 (1 -2): 69-83). Typically, cells contain only one type of mitochondrial DNA (mtDNA), which is called homoplasmy, but heteroplasmy does exist, usually in the form of a combination of mutant mtDNA and wild-type mtDNA molecules, or forms a combination of wild-type variants (Spikings et al., Hum. Repro. Update (2006) 12 (4): 401-415). Heteroplasmia can lead to mitochondrial disease; There are various mechanisms guaranteeing only maternal transmission. However, with increasing use of protocols that bypass the normal mechanisms for maintaining homoplasma (e.g., cytoplasmic transfer (CT) and SCNT), impaired mitochondrial function can occur in stem cells derived from these sources.

In one aspect, since parthenotes have a single parent, the likelihood of heteroplasmy is minimized.

Other diseases and conditions that can be treated using cell therapy include, for example, spinal cord injuries, multiple sclerosis, muscle dystrophy, diabetes, liver diseases, including acute diseases (viral hepatitis, drug overdoses (acetaminophen) and others), chronic diseases (chronic hepatitis and others (usually leading to cirrhosis), hereditary liver defects (hemophilia B, factor IX deficiency, bilirubin metabolism defects, urea cycle defects, lysosomal disease accumulation, A1-antitrypsin deficiency and others), heart disease, cartilage replacement, burns, foot ulcers, gastrointestinal diseases, vascular diseases, kidney disease, retinal disease, urinary tract disease and diseases and conditions associated with aging.

This methodology can be used to replace defective genes, for example, defective genes of the immune system, cystic fibrosis genes, or to introduce genes that result in the expression of therapeutically favorable proteins, such as growth factors, lymphokines, cytokines, enzymes, etc.

For example, a gene encoding a brain derived growth factor can be introduced into human pluripotent cells made in accordance with the present invention, cells differentiated into nerve cells, and cells transplanted into a patient with Parkinson's disease to slow down the loss of nerve cells during said diseases.

Also, pluripotent human ES cells of a subject can be used as an in vitro model of differentiation, in particular, to study genes that are involved in clinical and biological studies. These studies include, but are not limited to, the regulation of early development, regenerative medicine, or drug development. For example, the insertion of reporter genes (e.g., green fluorescent protein (gfp), luciferase, etc.) that are transcribed with the genes of interest can be used to study disease, developmental biology, regenerative medicine, or drug development. Also, differentiated cells, tissues, and organs obtained using the subject's ES cells can be used to study drugs.

In addition, the subject's ES cells, or differentiated cells derived from them, can be used as nuclear donors to produce other ES cells and cell colonies.

In addition, pluripotent cells obtained in accordance with the present description, can be used to identify proteins and genes that are involved in embryogenesis. This can be accomplished, for example, by differential expression, i.e. comparing mRNAs that are expressed in pluripotent cells obtained in accordance with the present invention with mRNAs that are expressed as these cells differentiate into various cell types, for example nerve cells, cardiomyocytes, other muscle cells, skin cells, and the like. Thus, it is possible to determine which genes are involved in the differentiation of specific cell types.

In addition, ES cells and / or their differentiated offspring that have specific genetic defects, such as a genetic defect leading to Duchenne muscular atrophy, can be used as models to study a specific disease associated with a genetic defect.

Another objective of the present description is the impact on pluripotent cell lines obtained in accordance with the described methods, mixtures of various growth factors, at different concentrations and under different conditions of cell cultivation, such as cultivation on different cell matrices or at different partial pressures of gases to identify conditions that induce the production and proliferation of the desired differentiated cell types.

The following examples are intended to illustrate, but not limit the invention.

EXAMPLES

Obtaining parthenogenetic human embryonic stem cells

Materials and methods

Selection of donors and the process of obtaining donor consent based on the information received

Donors were recruited from a group of women who first contacted the IVF center and were deemed suitable for the IVF procedure in accordance with clinical requirements.

Each potential donor was contacted by her attending physician and informed and advised on issues related to the study. If the donor made a decision on participation, she was presented with the correct document on consent based on the information received (studied and approved by the independent US-based ESCRO Committee), written in Russian, which outlined the objectives and procedures of the study. If a potential donor had questions, the doctor was at his disposal. Only potential donors who signed up consent based on the information received participated in the study.

Donors provided donor oocytes voluntarily and free of charge. The signed consent, based on the information received, said that all donor material was intended for research, and not for reproductive purposes, namely for developing methods for producing human ES cells and their differentiated descendants.

Suitability for research was determined in accordance with the FDA rules for determining the suitability of donors of human cells, tissues and products based on cells and tissues (FDA HCT / Ps, 2004), as well as in accordance with Order No. 67 (02.26.2003) of the Ministry of Health of the Russian Federation . This included a thorough medical examination with chest x-ray, blood tests (including liver function tests), and urine tests. Screening was also performed on Chlamydia trachomatis, Neisseria gonorrhoeae, syphilis, HIV, HBV and HCV.

Potential donors and their parents were further selected to participate in the study according to the HLA type.

In this protocol, the priority for oocyte collection was a successful IVF procedure. The best, fully developed cell-oocyte complexes were selected for IVF. If the total number of collected oocytes was less than 11, the woman was automatically expelled from the donor for research purposes.

Superovulation in donors

Each donor underwent ovarian stimulation using FSH (Gonal-F, Lab. Serono, Switzerland) during the period from the 3rd to the 13th day of the menstrual cycle. A total of 1,500 IU was administered. From the 10th to the 14th day of the donor’s menstrual cycle, a gonadoliberin antagonist Orgalutran (Organon, Holland) was injected at a dose of 0.25 mg per day. From the 12th to the 14th day of the donor menstrual cycle, 75 IU FSH + 75 IU LH were daily injected (Menopur, Ferring GmbH, Germany). If ultrasound revealed follicles with a diameter of 18 to 20 mm, a single dose of 8000 IU hCG (Choragon, Ferring GmbH, Germany) was administered on the 14th day of the donor's menstrual cycle. Transvaginal puncture was performed 35 hours after hCG injection, approximately on the 16th day. Under general anesthesia, donors collected follicular fluid from antral follicles by aspiration using a needle under ultrasound control into sterile tubes.

Oocyte activation and cultivation of parthenogenetic embryos

Cell-oocyte complexes (SOS) were removed from the follicular fluid, washed in a washing medium (MediCult), and then incubated in a universal medium for IVF (MediCult) with a coating layer of liquid paraffin (MediCult) for two hours in a humidified atmosphere containing 20% O ₂ , 5% CO ₂ at 37 ° C. Prior to activation, the SOS was treated with Syn Vitro hyadase (MediCult) to remove cumulative cells, followed by incubation in universal IVF medium with a coating layer of liquid paraffin for 30 minutes. Further cultivation of oocytes and embryos was carried out in a humidified atmosphere at 37 ° C in a gas mixture with a low content of O ₂ (90% N ₂ + 5% O ₂ + 5% CO ₂ ), with the exception of treatment A23187, which was carried out under the conditions described for SOS culture. Activation was carried out in a universal medium for IVF with a coating layer of petroleum jelly by sequentially exposing the oocytes to 5 μM A23187 (Sigma) for five minutes and 10 μg / ml of puromycin (Sigma) or 1 mm 6-DMAP (Sigma) for four hours followed by thorough washing of the oocytes in a universal medium for IVF. The oocytes were then placed in fresh IVF medium with a coating layer of liquid paraffin for subsequent cultivation. The next day (day 1), parthenogenetically activated oocytes were cultured to the blastocyst stage using sequential BlastAssist System media (MediCult) according to the manufacturer's recommendations. From the obtained blastocysts, internal cell mass (ICM) was isolated on the fifth to sixth day of cultivation.

Isolation of the inner cell mass of the blastocyst and cultivation of hpSC-Hhom

Zona pellucida was removed by treatment with 0.5% pronase (Sigma). Whole blastocysts were placed on the feeding layer of human neonatal fibroblasts (NSFs) mitotically inactivated with mitomycin C (Revazova et al., 2007, supra) in a medium developed for culturing hpSC-Hhom. When trophoblast cells are distributed over the surface after blastocyst attachment, ICM becomes visible. After three to four days of additional cultivation, the ICMs were isolated by mechanical sections of the ICM from the outgrowth of the trophoblast using a thinly elongated glass pipette. Isolated ICM was placed on a fresh nutrient layer and cultured for an additional three to four days. The first colony was mechanically cut and re-placed on the medium after five days of cultivation. All subsequent passages were carried out after five to six days of cultivation. The colonies of the early passages were mechanically divided into separate clusters of cells and re-placed on the medium. Then, hpSC-Hhom passages were performed using collagenase IV treatment and mechanical dissociation. Propagation of hpSC-Hhom was carried out at 37 ° C, 5% CO ₂ in a humidified atmosphere.

Applicants used VitroHES (Vitrolife) supplemented with 4 ng / ml hrbFGF (Chemicon), 5 ng / ml hrLIF (Chemicon) and 10% human umbilical cord blood to cultivate ICM and hpSC-Hhom. The NSF culture medium consisted of 90% DMEM (high glucose, with L-glutamine) (Invitrogen), 10% human umbilical cord blood serum and penicillin-streptomycin (100 U / 100 μg) (Invitrogen). Prior to the manufacture of the medium, human serum umbilical cord blood serum was screened for syphilis, HIV, HBV and HCV.

Characterization of hpSC-Hhom

For staining on embryonic stem cell markers using an immune tag, hpSC-Hhom colonies were fixed at room temperature for 20 minutes using 4% paraformaldehyde to identify SSEA-1, SSEA-3 and SSEA-4; 100% methanol was used for five minutes at minus 20 ° C to identify the remaining markers. Monoclonal antibodies used included: SSEA-1 (MAB4301), SSEA-3 (MAB4303), SSEA-4 (MAB4304), TRA-1-60 (MAB4360) and TRA-1-81 (MAB4381) from Chemicon, as well as OCT- 4 (sc-9081) from Santa Cruz Biotechnology; secondary antibodies included Alexa Fluor 546 (orange fluorescent) and 488 (green fluorescent) from Molecular Probes (Invitrogen). The nuclei were stained with DAPI (Sigma). Alkaline phosphatase and telomerase activity was determined using the AR kit and the TRAPEZE kit (Chemicon). Slices of chromosomes were made in the usual way. Giemsa disk karyotyping was performed according to the trypsin-Giemsa technique, and 30-100 metaphases were karyotyped in each case.

Embryoid Taurus Formation and Neural Differentiation

HpSC-Hhom colonies were mechanically divided into separate cell clusters and re-placed on 24-cell cluster plates pre-coated with 2% agarose (Sigma) in medium containing 85% knockout DMEM, 15% human umbilical cord blood serum, 1xMEM NEAA, 1 mM Glutamax, 0.055 mM P-mercaptoethanol, penicillin-streptomycin (50 U / 59 μg) (all from Invitrogen, excluding serum). Embryoid bodies were cultured in suspension for 14 days, followed by inoculation on plates for development of outgrowth or additional cultivation in suspension for one week.

Neural differentiation was induced by culturing two-week-old embryoid bodies attached to the surface of the culture dish for one week in differentiation medium DMEM / F2, B27, 2 mM Glutamax, penicillin-streptomycin (100 U / 100 μg) and 20 ng / ml hrbFGF (all from Invitrogen). All embryoid bodies gave rise to differentiated cells with neural morphology, others were dissected and further cultured to obtain neurospheres.

Pulsating embryoid bodies appeared spontaneously after five days of cultivation after being placed on the adhesive surface in the same medium that was used to generate the embryoid bodies.

Immunohistochemistry of differentiated derivatives of hpSC-Hhom

Embryoid bodies, neurospheres, or contracting embryoid bodies were placed on micro-cover glasses (VWR Scientific Inc.) coated with poly-D-lysine (Sigma) and cultured for approximately one week in a medium suitable for differentiation. For immune staining, differentiated cells were fixed with 100% methanol for five minutes at minus 20 ° C.

To identify ectodermal markers, we used a monoclonal mouse anti-neurofilament antibody 68 (Sigma), anti-human CD56 (NCAM) antibody (Chemicon) and anti-tubulin beta III (Chemicon) to detect neuronal markers. Antibody (Chemicon) against glial fibrillar acid protein (GFAP) was used to detect a glial cell marker.

To detect mesodermal markers in three-week embryoid bodies or contracting embryoid bodies, a monoclonal mouse anti-desmin antibody (Chemicon), an anti-human alpha actinin antibody (Chemicon) as muscle specific markers were used, and an anti-human CD31 / PECAM- ¹ antibody (R&D Systems) and an anti-human VE-cadaverin antibody (CD 144) (R&D Systems) were used as endothelial markers.

A monoclonal mouse antibody against human alpha-fetoprotein (R&D Systems) was used to identify endodermal markers in embryoid bodies. Secondary antibodies Alexa Fluor 546 (orange fluorescent) and 488 (green fluorescent) were obtained from Molecular Probes (Invitrogen). The nuclei were stained with DAPI (Sigma). NLA is genotyped.

The study of HLA haplotypes and HLA genotyping was carried out in donors and their parents. Genomic DNA was extracted from blood, cumulative cells, hpSC-Hhom and NSF using Dynabeads DNA Direct Blood from Dynal (Invitrogen). HLA genotyping was performed using PCR with an allele-specific sequencing primer (PCR-SSP, Protrans). All tests were performed in accordance with the manufacturer's instructions.

Asymmetric SNP Microarray Analysis

Genomic DNA was isolated from blood, cumulative cells, hpSC-Hhom and NSF using the phenol / chloroform extraction method. DNA samples obtained from three donors, four hpSC-Hhom and NSF lines were genotyped using Affymetrix Mapping 250K Nsp Arrays. Since the initial data series, containing 252973 binary SNP markers, exceeded the amount needed to determine the equivalence of genomic samples, it was reduced to simplify the calculations.

The following criteria were used to select markers based on genetic considerations: 1) the higher the degree of heterozygosity in the markers, the more information they provide to identify the origin of SNP samples. Heterozygosity of binary SNP markers is capped at a maximum of 0.5. Only SNP markers with a heterozygosity greater than 0.375 were selected (i.e., no alleles having a frequency of less than 0.25 or more than 0.75 in the white race population); 2) used all 22 autosomal chromosomes; 3) markers with low reliability were removed, since the identification of samples of common origin is very sensitive to genotyping errors. In the Affymetrix dataset, high confidence scores correspond to low reliability, so markers with high confidence scores were removed. In the standard setting, the marker request was not set if its confidence rating score exceeded 0.25. Even more stringent requirements were imposed on reliability by using only those markers that had confidence scores of less than or equal to 0.02 for all 12 samples.

Using these criteria, the number of SNP markers was reduced from 252973 to 4444. One final step was taken to reduce the number of markers (random sampling was not performed) by selecting only those with a distance between the markers of at least 0.1 cm (1 Mbp = 1 cM), because markers located very close to each other provide less information due to the close relationship between them. These steps led to the final selection of 3993 markers.

The 3993 markers thus selected were analyzed using Relcheck (version 0.67, copyright (C) 2000 Karl W. Broman, Johns Hopkins University, licensed as General Public License version 2 (June 1991)). Relcheck provides a way to determine the relationship between a pair of SNP samples. This is based on the calculation of the likelihood ratio when observing a given marker configuration in accordance with the genetic relationships of the samples. The use of Relcheck is based on the knowledge that since monozygotic twins have the same DNA, an equivalence test between two samples can be performed by checking whether these samples can be derived from monozygotic twins. Relcheck was carried out under the assumption that the genotyping error rates were approximately 0.4%. The Relcheck program identifies five types of relationships: monozygotic twins, a parent / offspring, siblings (full siblings), half-siblings or unmarried siblings (half-siblings) and non-relatives (Boehnke M. et al., Am. J. Hum. Genet . (1997) 61: 423-429; Broman KW et al., Am. J. Hum. Genet. (1998) 63: 1563-1563).

In order to determine the proportion of heterozygous SNPs in 15 chromosomes for the donor and stem cell samples, 1,495 randomly selected SNP markers used for earlier analysis were used, analyzed using 6 stem cell lines, 4 donors, and 1 control sample.

The location of the centromere for each chromosome was determined by taking the midpoint between the last available p-marker (short arm) and the first available q-marker (long arm) for each of chromosomes 1-12. For acrocentric chromosomes 13–15, there were no SNP markers on the p-arm; therefore, the centromere was considered to be located at position 0 (the beginning of the q-arm).

The following values were used as the centromere position for chr 1-15 (in Mbp from the end of the p-arm):

Chromosome No. Mbp chr1 131.5051 Chr2 92.7926 Chr3 93.1514 Chr4 51,0889 Chr5 46.8726 Chr6 60.5099 Chr7 57,8019 Chr8 45.3089 Chr9 54,6881 chr10 39.0399 Chr11 52,7549 Chr12 35,6473 Chr13 0 Chr14 0 Chr15 0

For each SNP marker, the absolute distance from the centromere on a particular chromosome was calculated (that is, both directions from the centromere were treated equivalently when all “negative” distances were converted to positive distances). For the purpose of calculating the proportion of heterozygotes, the distances of the chromosomes (from centromeres) were divided into 15 intervals (called “bays” at the exit). Each interval consisted of 10 Mbp in length. The number assigned to each “compartment” corresponded to the upper boundary at the chromosome distance for a given interval. All units were expressed in mega base pairs (Mbp):

For example:

Compartments = 10 correspond to the interval [0.10] Mbp from centromeres.

Compartments = 70 correspond to the interval [60.70] Mbp from the centromeres.

Compartments = 150 correspond to the interval [140,150] Mbp from the centromeres.

Base pair distances provided in the Affymetrix SNP dataset were used to maintain consistency with previous data analysis. For each interval, the proportion of heterozygosity was estimated using the average value of indicator variables for heterozygosity for all SNPs within the interval.

For the donor and stem cells, the heterozygosity variable stores the average value of indicator variables for heterozygosity at each location of the SNP marker for the donor (2, 12, 10, 11) and stem cell samples (3, 4, 5, 9, 6, 7, 8 ) The corresponding values for fractions of heterozygosity can be easily obtained by subtracting the values of heterozygosity from 1.

Internal controls correctly identified the paired genotypic relationship between split cultures derived from the same hpSC-Hhom line as “monozygotic twins”.

Imprinting Gene Analysis

Total RNA was extracted as described (Chomcznski and Sacchi, Anal. Biochem. (1987) 162: 156-159) and precipitated with isopropanol. The remaining genomic DNA was removed using RNase-free DNAse treatment (Promega). cDNA was synthesized from 1 μg of total RNA using RevertAid M-Mul V (Fermentas) reverse transcriptase in 20 μl of reaction volume. PCR reactions were carried out with 1 μl of cDNA using Taq DNA polymerase (Fermentas). All reactions were carried out in accordance with the manufacturer's instructions.

The sequence of primers and PCR conditions were as follows: TSSC5 (Lee et al., Cancer Res. (1998) 58: 4155-4159) forward primer 5'-GCTCTTCATGGTCATGTTCTCCA-3 '(SEQ ID NO: 1) and reversible primer 5'- GGAGCAGTGGTTGTACAGAGG-3 '(SEQ ID NO: 2), at 94 ° C. for 4 minutes in an amount of 1 cycle; 94 ° C for 1 min, 55 ° C for 1 min and 72 ° C for 1 min in an amount of 33 cycles. The size of the product was 364 bp. H19 (Hashimoto et al., Nat. Genet. (1995) 9: 109-110) 5'-TACAACACTGCACTACCTG-3 'forward primer (SEQ ID NO: 3) and 5'-TGGCCATGAAGATGGAGTCG-3' reverse primer (SEQ ID NO: 4), under conditions of 94 ° C for 4 min in an amount of 1 cycle; 94 ° C for 1 min, 52 ° C for 1 min and 72 ° C for 1 min in an amount of 38 cycles. The size of the product was 148 bp. PEG_1 (Li et al., J. Biol. Chem. (2002) 277: 13518-13527) 5'-GAG TCC TGT AGG CAA GGT CTT ACC T-3 'forward primer (SEQ ID NO: 5) and reverse primer 5'-CTT GCC TGA AGA CTT CCA TGA GTG A-3 '(SEQ ID NO: 6), under conditions of 94 ° C for 4 min in an amount of 1 cycle; 94 ° C for 1 min, 55 ° C for 1 min and 72 ° C for 1 min in an amount of 35 cycles. The size of the product was 155 bp. PEG_2 (Li et al., Supra) forward primer 5'-GCT GCT GGC CAG CTC TGC ACG GCT G-3 '(SEQ ID NO: 7) and reversible primer 5'-CTT GCC TGA AGA CTT CCA TGA GTG A- 3 '(SEQ ID NO: 8), under conditions of 94 ° C for 4 min in an amount of 1 cycle; 94 ° C for 1 min, 65 ° C for 1 min and 72 ° C for 1 min in an amount of 39 cycles. The size of the product was 230 bp. SNRPN (Glenn et al., Hum. Mol. Genet. (1993) 2: 2001-2005) 5'-CTTAGCTGAGACACCAAGAGG forward primer (SEQ ID NO: 9) and 5'-GCAGCATCTTTGCTACTCTTGC-3 'reverse primer (SEQ ID NO : 10), under conditions of 94 ° C for 4 min in an amount of 1 cycle; 94 ° C for 1 min, 55 ° C for 1 min and 72 ° C for 1 min in an amount of 33 cycles. The size of the product was 246 bp. GAPDH (Adjaye et al., Gene (1999) 237: 373-383) 5'-ACCACAGTCCATGCCATCAC forward primer (SEQ ID NO: 11) and 5'-TCCACCACCCTGTTGCTGTA-3 'reverse primer (SEQ ID NO: 12) under conditions 94 ° C for 4 min in an amount of 1 cycle; 94 ° C for 1 min, 55 ° C for 1 min and 72 ° C for 1 min in an amount of 21 cycles. The product size was 450 bp.

PCR products were analyzed using 5% polyacrylamide gel electrophoresis (5 μl per line), stained with ethidium bromide and documented using a Biolmaging (UVP) system. RT-PCR experiments were performed repeatedly with reproducible results. GAPDH served as an expressed control throughout. Genomic contamination was excluded by including an RT-negative sample (without reverse transcriptase at the reverse transcriptase stage) in each PCR series as a control.

Teratoma Education and Assessment

All animal procedures were carried out by the following institutions: Biological Testing Laboratory-Branch of Shemyakin and the Institute of Bioorganic Chemistry of the Russian Academy of Sciences Ovchinnikov (Pushchino, Moscow Region, Russia), accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

SCID immunodeficient mice that were hatched from CB17 / ICR-PRKDC-SCID CRL mice obtained from Charles River Laboratories Research Model and Services (Germany) were used to monitor teratoma formation. For injection, hpSC-Hhom mice were enzymatically separated from culture plates using type IV collagenase and then resuspended to form clusters of cells. About two to five million hpSC-Hhom cells were injected subcutaneously into the hind limb. About two months later, the resulting teratomas were removed and fixed with 4% paraformaldehyde. Half of the tissue was cryoprotected with sucrose, and the other half was poured with 5% agar-agar, and then 60 μm sections were made using a microtome. Sections were placed on coverslips and stained with hematoxylin-eosin according to Kraberg, Van Gieson and picrofuscin.

About two to five million human mitomycin C-treated fibroblasts used as supply layers for phEC cells were injected as controls. In control animals, teratom growth was not observed.

Example 1. Parthenot generation

Five oocyte donors, all older than 31 years old, participated in this study. Oocytes were obtained using hormonal stimulation with the main intention of in vitro fertilization (IVF). A total of 46 cell-oocyte complexes (SOS) were selected from five donors and used in this study (table 1).

Table 1
Parthenot generation and parthenogenetic embryonic stem cell lines Donor Received Oocytes Donated Oocytes Activated oocytes Created Partenotes ⁵ Received blastocysts ⁶ Generated lines The fate of the donor with ICM without visible ICM one 8 four four four 2 - Immunosurgery phESC-1 pregnant 2 fifteen 8 8 8 3 3 phESC-3
phESC-4
phESC-5
all from whole blastocysts pregnant with twins 3 27 fourteen 14 ^l ' ^a eleven 3 2 phESC-6
from whole blastocyst pregnant four 22 eleven 11 ³ 10 2 3 phESC-7
from whole blastocyst pregnant 5 twenty 9 ⁴ 7 7 one four cell line not generated not pregnant ¹ - two oocytes were not activated; ² - one oocyte degenerated after activation; ³ - one oocyte was not activated; ⁴ - two oocytes were at the stage of metaphase I and were excluded; ⁵ - whole parthenogenetic oocytes = 40; ⁶ - total blastocysts obtained = 23

Before activation of the oocytes, the SOS was kept at atmospheric oxygen tension. After the removal of the cumulative cells, only normal oocytes in metaphase II with a distinct first polar body were taken for the activation procedure. Oocytes were activated with 5 mM ionomycin for five minutes, followed by incubation with 6-DMAP for four hours to prevent expulsion of the second polar body and diploid embryo production. Oocytes and embryos were manipulated and cultured in Medical medium in accordance with the manufacturer's instructions using standard IVF procedures and under reduced oxygen tension (90% N ₂ + 5% O ₂ + 5% CO ₂ ). Only 40 oocytes were able to cleave after parthenogenetic activation. These procedures allowed the production of 23 blastocysts on the fifth or sixth day of embryo cultivation. Eleven blastocysts had visible ICM (table 1).

Differentiation of parthenogenetic lines of hESC

It is very important to minimize, if not eliminate, the components of animal origin during differentiation and cultivation of hESC intended for clinical use. For this purpose, some researchers do not use immunosurgery to isolate ICM, but mechanical means to isolate from whole blastocysts (Kim et al., Stem Cells (2005) 23: 1228-1233). Culture media and nourishing cells may also contain animal pathogens. To eliminate possible contamination, the researchers used human cells as a supply layer instead of murine fibroblasts (Cheng et al., Stem Cells (2003) 21: 131-142; Hovatta et al., Hum. Repro. (2003) 18: 1404-1429; Stojkovic et al., Stem Cells (2005) 23: 306-314) or were cultured with hESC in the absence of a supply layer and serum (Amit et al., 2004; Klimanskaya et al., Lancet (2005) 365: 1636-1641). Taking into account the above previous studies, the culture conditions for the isolation of ICM and phESC have been modified.

For the differentiation and cultivation of phESC, human neonatal skin fibroblasts (NSF) mitotically inactivated mitomycin C were used as feeding cells. These cells were originally obtained and propagated using a medium containing human umbilical cord blood serum instead of animal serum. The culture medium for phESC consisted of VitroHES medium (Vitrolife) supplemented with human serum obtained from umbilical cord blood, hrbFGF and hrLIF. phESC was propagated at 37 ° C, 5% CO ₂ , humidified atmosphere.

All parthenogenetic blastocysts obtained were first treated with 0.5% pronase to remove zona pellucida. Well-formed ICMs from two blastocysts were obtained from the first oocyte donor using trypsin treatment (Li et al., Mol. Reprod. Dev. (2003) 65: 429-434) and conventional immunosurgery (Solter and Knowles, Proc. Natl. Acad. Sci. USA (1975) 72: 5099-5102). ICMs were then placed on human feeder cells under the conditions described for phESC production. ICM from trypsin-treated blastocysts did not produce living cells. The ICM obtained after immunosurgery demonstrated cell growth, resulting in the creation of a phESC-1 cell line. The remaining 21 blastocysts were first placed on the supply layer under the described conditions. ICM were isolated by mechanical sections from the growth of trophoblast cells and re-placed on fresh feeder cells. Five phESC lines (from phESC-3 to phESC-7) were generated by this method (table 1).

Characterization of phESC lines

The phESC lines demonstrate the morphology that was expected from hESC and form colonies from tightly packed cells, with noticeable nucleoli and a small ratio of cytoplasm and nucleus (Figure 1). These cells express traditional hES markers of SSEA-3, SSEA-4 (Figure 1), TRA-1-60, TRA-1-81 and OST-4 cells (Figure 1, continued) and do not express SSEA-1, a positive marker for undifferentiated murine embryonic stem cells (figure 1). Cells obtained from all lines show high levels of alkaline phosphatase activity (Figure 1, continued) and telomerase (Figure 2).

Giemsa disk karyotyping showed that the phESC lines have a normal human 46, XX karyotype, with the exception of the phESC-7 line (figure 3). Approximately 91% of cells from the phESC-7 line have a karyotype of 47, XXX, and 9% of the cells have a karyotype of 48, XXX, + 6. A different degree of X chromosome heteromorphism was observed when analyzing 100 metaphases in cell lines: approximately 12% of the cells in phESC-1 and phESC-6 lines showed X chromosome heteromorphism; 42% of the cells in the phESC-5 and 70% lines, 80% and 86% in the phESC-7, phESC-3 and phESC-4 lines, respectively, showed heteromorphism of the X chromosome (Figure 3).

The ability of phESC to differentiate

The phESC-1 line remained undifferentiated for 10 months of cultivation with 35 passages. Other cell lines were successfully cultured for at least 21 passages. Cells from all phESC lines formed cystic embryoid bodies in suspension culture and gave rise to derivatives of all three germ layers, ectoderm, mesoderm, and endoderm after in vitro differentiation (Figure 4). Approximately 5% of phESC-1 embryoid bodies gave rise to pulsating cells five days after plating. The phESC-6 line produced pigmented epithelium-like cells (Figure 4 L, K). Ectodermal differentiation is represented by positive immunocytochemical staining for neuron-specific markers neurofilament 68 (Figure 4A), NCAM (Figure 4B), tubulin beta-III (Figure 4C) and the glial cell marker GFAP (Figure 4D, M). Differentiated cells were positive for mesodermal markers, including alpha actinin (Figure 4G) and desmin (Figure 4J), which are muscle-specific markers, and endothelial markers PEC AM-1 (Figure 4E) and VE-cadherin (Figure 4F) ) Endodermal differentiation is represented by positive staining of the differentiated derivatives on alpha-fetoprotein (Figure 4H, L). The ability of phESC lines to form derivatives of all three germ layers was studied in vivo by subcutaneous injection of phESC in mice and rats with immunodeficiency (Figure 5). Cells from all phESC lines were able to form teratomas approximately two months after injection. Histological examination revealed organized structures, including: epithelium, capsule, smooth muscle, adipose tissue, hematogenous tissue, neural tube, and glandular epithelium. Immunohistochemical analysis revealed a positive staining for beta-tubulin (Figure 5C) - ectoderm marker; fibronectin (Figure 5B) and muscle actin (Figure 5A) are mesodermal markers; and alpha-fetoprotein (Figure 5D) is an endodermal marker. These data demonstrate that phESC can differentiate in vivo into all three germ layers, which lead to the formation of all cell types in the human body.

DNA profile analysis in phESC lines

Comparative analysis of the DNA profile was carried out in all phESC lines, somatic donor cells and feeder cells. In these studies used analysis affymetrix single nucleotide (SNP) polymorphisms (Mapping 50K Hind 240 Arrays) to confirm the genetic similarity of phESC to somatic donor cells. A total of 1459 SNP markers along 15 autosomes (chromosomes 1-15) were selected with an average distance between markers of 1.12 Mbp. All paired genotypic relationships between phESC lines and their corresponding somatic donor cells were identified as “complete siblings” (genetically matched), and all other combinations of pairs were identified as “unrelated”. Internal controls identified paired genotypic relationships between separated cultures derived from the same phESC line as “monozygotic twins (Table 2).

The analyzed DNA samples were numbered as follows: 1 - human neonatal skin fibroblasts, 2 - phESC-7 line donor, 3 - phESC-7 line, 4 - phESC-1 line, 5 - phESC-1 line, 6 - phESC-3 line 7 - phESC-4 line; 8 - phESC-5 line; 9 - phESC-6 line; 10 - phESC-6 line donor; 11 - phESC-3 to phESC-5 line donor; 12 - phESC-1 line donor .

The IBS columns at the output show the number of markers by which pairs were typed, and have common 0, 1, or 2 alleles that are identical in state. (For MOH twins under ideal conditions of no genotyping errors, all markers should be placed under IBS = 2.) The output does not show P (observed markers I relationship data) directly, but it shows the sum of points LOD = log ₁₀ {P (observed markers I estimated relationship ) / P (observed markers of I relationship for which the maximum probability was obtained and, thus, a request was made)} as a measure of similarity. The lower the total LOD score, the less likely the expected relationship between the two samples is.

A comparative analysis of SNP markers showed that, in general, donor cells do not appear to show a clear pattern of heterozygosity tendency along distances from centromeres, while stem cells show slightly smaller heterozygosity fractions near centromeres and telomeres compared to heterozygosity fractions in the middle as a result of possible recombination of chromosomes (tables AI).

Table a
Chromosomal compartments of a heterozygous donor / stem cell Chromosomal compartment of heterozygous donor / stem cells Chromosome Compartments Frequency Heterozygous donor Heterozygous stem cell 1459 0.37 0,207 10 91 0.407 0,053 twenty 135 0.33 0,111 thirty 185 0,358 0.19 40 169 0.408 0.285 fifty 157 0.385 0.268 60 153 0.353 0.204 70 123 0.354 0.188 80 113 0.345 0.239 90 118 0.371 0.228 one hundred 88 0.398 0.24 110 49 0.393 0.233 120 29th 0.302 0.222 130 26 0.365 0.231 140 twenty 0.4 0.193 150 3 0.5 0,048 one 142 0.373 0.198 2 150 0.327 0.187 3 111 0.41 0.229 four 133 0.346 0.179 5 121 0.39 0.263 6 115 0.387 0.205 7 103 0.379 0.205 8 93 0.401 0.187 9 66 0.314 0.171 10 88 0.375 0.273 eleven 78 0.385 0.19 12 77 0.403 0.161 13 78 0.324 0.192 fourteen 62 0.379 0.279 fifteen 42 0.345 0.194 one twenty 9 0.361 0,063 one thirty fourteen 0.357 0.153 one 40 12 0.354 0.214 one fifty fifteen 0.35 0.229 one 60 21 0.369 0.211 one 70 19 0.461 0.211 one 80 9 0.333 0.175 one 90 13 0.288 0.176 one one hundred 13 0.327 0.198 one 110 8 0.469 0.214 one 120 6 0.333 0.19 one 130 3 0.667 0.571 2 10 3 0.333 0 2 twenty eleven 0.386 0.182 2 thirty 13 0.269 0.176 2 40 6 0.25 0.167 2 fifty 19 0,303 0.263 2 60 13 0.423 0,308 2 70 10 0.275 0.171 2 80 13 0.327 0.176 2 90 19 0.342 0.226 2 one hundred 7 0.214 0,082 2 110 four 0.375 0.143 2 120 7 0.25 0.122 2 130 eleven 0.318 0.143 2 140 eleven 0.386 0.156 2 150 3 0.5 0,048 3 10 9 0.417 0.016 3 twenty ,fifteen 0.333 0,057 3 thirty 12 0.354 0.167 3 40 eleven 0.455 0.351 3 fifty 3 0,083 0.143 3 60 12 0.438 0.286 3 70 12 0.458 0.202 3 80 fourteen 0.5 0.347 3 90 12 0.375 0.25 3 one hundred 9 0.472 0.429 3 110 2 0.375 0.286 four 10 13 0.423 0.121 four twenty 9 0.25 0.143 four thirty fifteen 0.283 0.152 four 40 fifteen 0.367 0.2 four fifty eleven 0.409 0.195 four 60 8 0.375 0.179 four 70 7 0.286 0.163 four 80 9 0.278 0.206 four 90 9 0.389 0.222 four one hundred 7 0.321 0.204 four 110 8 0.375 0.196 four 120 7 0.321 0.184 four 130 8 0.313 0.179 four 140 7 0.429 0.204 5 10 12 0.417 0,083 5 twenty 12 0.292 0.119 5 thirty eleven 0.409 0.325 5 40 fourteen 0.411 0,306 5 fifty 10 0.45 0.257 5 60 8 0.219 0.107 5 70 8 0.344 0.25 5 80 8 0.406 0.339 5 90 8 0.375 0.214 5 one hundred 7 0.536 0.286 5 110 9 0.5 0.508 5 120 8 0.344 0.393 5 130 four 0.375 0.321 5 140 2 0.375 0.357 6 10 13 0.404 0,022 6 twenty fourteen 0.286 0,082 6 thirty eleven 0.364 0.208 6 40 12 0.542 0.488 6 fifty 10 0.4 0.329 6 60 eighteen 0.347 0.19 6 70 8 0.344 0.161 6 80 8 0.25 0.179 6 90 7 0.429 0.245 6 one hundred 10 0.55 0.229 6 110 four 0.313 0.143 7 10 6 0.292 0.024 7 twenty 12 0.333 0,071 7 thirty 16 0.313 0.143 7 40 fourteen 0.429 0.337 7 fifty 16 0.484 0.402 7 60 fifteen 0.433 0.171 7 70 9 0.389 0.19 7 80 5 0.3 0.171 7 90 7 0.214 0,061 7 one hundred 3 0.5 0.381 8 10 8 0.313 0,036 8 twenty 10 0.425 0.129 8 thirty fifteen 0.483 0.162 8 40 13 0.346 0.253 8 fifty eleven 0.364 0.208 8 60 four 0.25 0.143 8 70 10 0.45 0.314 8 80 6 0.375 0.167 8 90 eleven 0.455 0.169 8 one hundred 5 0.4 0.257 9 twenty 9 0.25 0.016 9 thirty fifteen 0.35 0.133 9 40 10 0.45 0.229 9 fifty 13 0.269 0.209 9 60 9 0.278 0.254 9 70 6 0.25 0.167 9 80 four 0.313 0.214 10 10 7 0.5 0.163 10 twenty fifteen 0.367 0.181 10 thirty 19 0.289 0.248 10 40 12 0.375 0.298 10 fifty 6 0.5 0.381 10 60 9 0.333 0.349 10 70 2 0.375 0.286 10 80 8 0.406 0.393 10 90 7 0.357 0.286 10 one hundred 3 0.5 0.238 eleven 10 7 0.357 0.02 eleven twenty 8 0.375 0.161 eleven thirty fifteen 0.45 0.267 eleven 40 fifteen 0.4 0.219 eleven fifty 16 0.469 0.268 eleven 60 7 0.143 0,061 eleven 70 6 0.333 0.119 eleven 80 four 0.313 0.179 12 10 13 0.481 0.011 12 twenty 9 0,306 0.143 12 thirty 13 0.385 0,132 12 40 12 0.458 0.226 12 fifty 9 0.5 0.238 12 60 5 0.25 0.114 12 70 7 0.214 0.163 12 80 3 0.333 0.333 12 90 four 0.563 0.393 12 one hundred 2 0.5 0,071 13 twenty one 0.25 0 13 thirty four 0.375 0.179 13 40 10 0.325 0.214 13 fifty 7 0.393 0,306 13 60 9 0.25 0,048 13 70 8 0.344 0.143 13 80 9 0.25 0.27 13 90 7 0.464 0.429 13 one hundred eleven 0.341 0.208 13 110 eleven 0.295 0,065 13 120 one 0 0 fourteen twenty one 0.25 0.143 fourteen thirty 7 0.429 0.265 fourteen 40 8 0.531 0.518 fourteen fifty 8 0.344 0.321 fourteen 60 9 0.444 0.27 fourteen 70 6 0.333 0.119 fourteen 80 8 0.219 0.125 fourteen 90 7 0.286 0.184 fourteen one hundred 6 0.417 0.333 fourteen 110 2 0.5 0.571 fifteen thirty 5 0.3 0.171 fifteen 40 5 0.4 0.286 fifteen fifty 3 0.167 0.143 fifteen 60 6 0.5 0.19 fifteen 70 5 0.1 0,086 fifteen 80 5 0.5 0.257 fifteen 90 7 0.429 0.245 fifteen one hundred 5 0.25 0.171 fifteen 110 one 0.25 0

Table B
Chromosome compartments, heterozygosity / frequency on chromosome 01 Hetero chromosome compartment 01 Chromosome Compartments Frequency Hetero 01 1459 0.402 10 91 0.429 twenty 135 0.393 thirty 185 0.357 40 169 0.438 fifty 157 0.376 60 153 0.425 70 123 0.407 80 113 0.442 90 118 0.373 one hundred 88 0.42 110 49 0.449 120 29th 0.448 130 26 0.346 140 twenty 0.25 150 3 0.333 one 142 0.352 2 150 0.373 3 111 0.45 four 133 0.391 5 121 0.347 6 115 0.435 7 103 0.427 8 93 0.409 9 66 0.439 10 88 0.341 eleven 78 0.436 12 77 0.39 13 78 0.474 fourteen 62 0.452 fifteen 42 0.405 twenty 9 0.333 thirty fourteen 0.429 40 12 0.167 fifty fifteen 0,067 60 21 0.333 70 19 0.579 80 9 0.333 90 13 0.538 one hundred 13 0,308 110 8 0.5 120 6 0.333 130 3 0 2 10 3 0.667 2 twenty eleven 0.273 2 thirty 13 0,308 2 40 6 0.5 2 fifty 19 0.421 2 60 13 0.462 2 70 10 0.3 2 80 13 0.538 2 90 19 0.263 2 one hundred 7 0.286 2 110 four 0.5 2 120 7 0.429 2 130 eleven 0.455 2 140 eleven 0.182 2 150 3 0.333 3 10 9 0.444 3 twenty fifteen 0.6 3 thirty 12 0.333 3 40 eleven 0.545 3 fifty 3 0.333 3 60 12 0.333 3 70 12 0.417 3 80 fourteen 0.357 3 90 12 0.5 3 one hundred 9 0.667 3 110 2 0 four 10 13 0.385 four twenty 9 0.333 four thirty fifteen 0.333 four 40 fifteen 0.467 four fifty eleven 0.273 four 60 8 0.5 four 70 7 0.286 four 80 9 0.556 four 90 9 0,111 four one hundred 7 0.571 four 110 8 0.625 four 120 7 0.429 four 130 8 0.375 four 140 7 0.286 5 10 12 0.417 5 twenty 12 0.25 5 thirty eleven 0.182 5 40 fourteen 0.357 5 fifty 10 0.1 5 60 8 0.625 5 70 8 0.25 5 80 8 0.625 5 90 8 0.375 5 one hundred 7 0 5 110 9 0.556 5 120 8 0.5 5 130 four 0.25 5 140 2 0.5 6 10 13 0.385 6 twenty fourteen 0.214 6 thirty eleven 0.636 6 40 12 0.5 6 fifty 10 0.3 6 60 eighteen 0.444 6 70 8 0.75 6 80 8 0.5 6 90 7 0.429 6 one hundred 10 0.3 6 110 four 0.5 7 10 6 0.833 7 twenty 12 0.417 7 thirty 16 0.188 7 40 fourteen 0.429 7 fifty 16 0.5 7 60 fifteen 0.467 7 70 9 0.333 7 80 5 0.4 7 90 7 0.429 7 one hundred 3 0.667 8 10 8 0.25 8 twenty 10 0.5 8 thirty fifteen 0.333 8 40 13 0.385 8 fifty eleven 0.364 8 60 four 0.5 8 70 10 0.5 8 80 6 0.5 8 90 eleven 0.364 8 one hundred 5 0.6 9 twenty 9 0.444 9 thirty fifteen 0.4 9 40 10 0.7 9 fifty 13 0.385 9 60 9 0.444 9 70 6 0.333 9 80 four 0.25 10 10 7 0.429 10 twenty fifteen 0.4 10 thirty 19 0.316 10 40 12 0.333 10 fifty 6 0.333 10 60 9 0.444 10 70 2 0 10 80 8 0.25 10 90 7 0.286 10 one hundred 3 0.333 eleven 10 7 0.429 eleven twenty 8 0.375 eleven thirty fifteen 0.533 eleven 40 fifteen 0.467 eleven fifty 16 0.438 eleven 60 7 0.286 eleven 70 6 0.5 eleven 80 four 0.25 12 10 13 0.385 12 twenty 9 0.556 12 thirty 13 0.231 12 40 12 0.5 12 fifty 9 0.444 12 60 5 0.4 12 70 7 0.429 12 80 3 0 12 90 four 0.25 12 one hundred 2 0.5 13 twenty one one 13 thirty four 0.5 13 40 10 0.5 13 fifty 7 0.714 13 60 9 0.333 13 70 8 0.125 13 80 9 0.667 13 90 7 0.571 13 one hundred eleven 0.545 13 110 eleven 0.273 13 120 one one fourteen twenty one 0 fourteen thirty 7 0.571 fourteen 40 8 0.25 fourteen fifty 8 0.5 fourteen 60 9 0.556 fourteen 70 6 0.333 fourteen 80 8 0.5 fourteen 90 7 0.429 fourteen one hundred 6 0.5 fourteen 110 2 0.5 fifteen thirty 5 0.2 fifteen 40 5 0.6 fifteen fifty 3 one fifteen 60 6 0.333 fifteen 70 5 0.4 fifteen 80 5 0.4 fifteen 90 7 0.286 fifteen one hundred 5 0.4 fifteen 110 one 0

Table C
Chromosome compartments, heterozygosity / frequency on chromosomes 02 and 03 Chromosome compartment hetero 02 03 Chromosome Compartments Frequency Hetero 02 Hetero 03 1459 0.408 0.215 10 91 0.407 0 twenty 135 0.37 0,096 thirty 185 0.389 0.141 40 169 0.444 0.26 fifty 157 0.484 0.35 60 153 0.366 0.235 70 123 0.423 0.26 80 113 0.381 0.265 90 118 0.458 0.314 one hundred 88 0.352 0.239 110 49 0.449 0.224 120 29th 0.31 0.138 130 26 0.346 0,038 140 twenty 0.4 0.15 150 3 0.333 0 one 142 0.423 0,07 2 150 0.38 0.18 3 111 0.486 0.261 four 133 0.398 0.173 5 121 0.405 0,306 6 115 0.443 0.278 7 103 0.447 0.165 8 93 0.43 0.28 9 66 0.333 0,076 10 88 0.375 0.295 eleven 78 0.423 0.218 12 77 0.403 0.234 13 78 0.282 0.218 fourteen 62 0.452 0.452 fifteen 42 0.381 0.024 one twenty 9 0.222 0 one thirty fourteen 0.429 0 one 40 12 0.417 0 one fifty fifteen 0.4 0 one 60 21 0.381 0 one 70 19 0.684 0 one 80 9 0.556 0,111 one 90 13 0.462 0.231 one one hundred 13 0.154 0,077 one 110 8 0.5 0.375 one 120 6 0.333 0.333 one 130 3 0.333 0 2 10 3 0.667 0 2 twenty eleven 0.364 0.273 2 thirty 13 0.231 0.154 2 40 6 0.667 0.5 2 fifty 19 0.316 0.316 2 60 13 0.231 0.231 2 70 10 0.4 0.4 2 80 13 0.385 0,077 2 90 19 0.421 0.263 2 one hundred 7 0.286 0 2 110 four one 0 2 120 7 0.286 0 2 130 eleven 0.364 0 2 140 eleven 0.455 0 2 150 3 0.333 0 3 10 9 0.556 0 3 twenty fifteen 0.4 0 3 thirty 12 0.333 0 3 40 eleven 0.455 0.364 3 fifty 3 0 0 3 60 12 0.667 0.583 3 70 12 0.583 0.25 3 80 fourteen 0.5 0.357 3 90 12 0.583 0.417 3 one hundred 9 0.444 0.444 3 110 2 0.5 0.5 four 10 13 0.385 0 four twenty 9 0.333 0,111 four thirty fifteen 0.333 0.133 four 40 fifteen 0.4 0.267 four fifty eleven 0.364 0.273 four 60 8 0.375 0.375 four 70 7 0.857 0.857 four 80 9 0.222 0.222 four 90 9 0.444 0 four one hundred 7 0.429 0 four 110 8 0.5 0 four 120 7 0.429 0 four 130 8 0.375 0 four 140 7 0.286 0.286 5 10 12 0.417 0 5 twenty 12 0.5 0.167 5 thirty eleven 0.455 0.364 5 40 fourteen 0.429 0.286 5 fifty 10 0.5 0.5 5 60 8 0.125 0.125 5 70 8 0.375 0.375 5 80 8 0.5 0.5 5 90 8 0.625 0.625 5 one hundred 7 0.286 0.286 5 110 9 0.333 0.333 5 120 8 0.25 0.25 5 130 four 0.25 0.25 5 140 2 0.5 0.5 6 10 13 0.538 0 6 twenty fourteen 0.5 0 6 thirty eleven 0.455 0 6 40 12 0.5 0.5 6 fifty 10 0.5 0.5 6 60 eighteen 0.389 0.389 6 70 8 0.375 0.375 6 80 8 0.25 0.25 6 90 7 0.429 0.429 6 one hundred 10 0.4 0.4 6 110 four 0.5 0.5 7 10 6 0.167 0 7 twenty 12 0.333 0 7 thirty 16 0.438 0.25 7 40 fourteen 0.429 0.143 7 fifty 16 0.688 0.438 7 60 fifteen 0.533 0.133 7 70 9 0.333 0 7 80 5 0.2 0 7 90 7 0.286 0 7 one hundred 3 one 0.667 8 10 8 0.25 0 8 twenty 10 0.4 0.2 8 thirty fifteen 0.467 0.267 8 40 13 0.462 0.231 8 fifty eleven 0.636 0.273 8 60 four 0.25 0.25 8 70 10 0.4 0.4 8 80 6 0.5 0.5 8 90 eleven 0.455 0.455 8 one hundred 5 0.2 0.2 9 twenty 9 0.333 0 9 thirty fifteen 0.467 0 9 40 10 0.5 0 9 fifty 13 0.231 0,077 9 60 -9 0.222 0.222 9 70 6 0.167 0.167 9 80 four 0.25 0.25 10 10 7 0.286 0 10 twenty fifteen 0.333 0.2 10 thirty 19 0.158 0,053 10 40 12 0.5 0.417 10 fifty 6 0.667 0.667 10 60 9 0.333 0.333 10 70 2 0.5 0.5 10 80 8 0.5 0.5 10 90 7 0.286 0.286 10 one hundred 3 one one eleven 10 7 0.143 0 eleven twenty 8 0.25 0.125 eleven thirty fifteen 0.6 0.2 eleven 40 fifteen 0.6 0.267 eleven fifty 16 0.625 0.438 eleven 60 7 0.143 0.143 eleven 70 6 0 0 eleven 80 four 0.25 0.25 12 10 13 0.538 0 12 twenty 9 0.333 0 12 thirty 13 0.462 0.231 12 40 12 0.167 0.167 12 fifty 9 0.778 0.778 12 60 5 0.2 0.2 12 70 7 0.286 0.286 12 80 3 0.333 0.333 12 90 four 0.5 0.5 12 one hundred 2 0 0 13 twenty one 0 0 13 thirty four 0.5 0.25 13 40 10 0.4 0.4 13 fifty 7 0.429 0.429 13 60 9 0 0 13 70 8 0.25 0.25 13 80 9 0.222 0.222 13 90 7 0.571 0.571 13 one hundred eleven 0.273 0,091 13 110 eleven 0.182 0 13 120 one 0 0 fourteen twenty one one one fourteen thirty 7 0.286 0.286 fourteen 40 8 0.375 0.375 fourteen fifty 8 0.5 0.5 fourteen 60 9 0.556 0.556 fourteen 70 6 0.5 0.5 fourteen 80 8 0.375 0.375 fourteen 90 7 0.429 0.429 fourteen one hundred 6 0.333 0.333 fourteen 110 2 one one fifteen thirty 5 0.2 0 fifteen 40 5 0.4 0 fifteen fifty 3 0.333 0 fifteen 60 6 0.833 0 fifteen 70 5 0 0 fifteen 80 5 0.4 0 fifteen 90 7 0.429 0 fifteen one hundred 5 0.4 0.2 fifteen 110 one 0 0

Table D
Chromosome compartments, heterozygosity / frequency on chromosomes 12 and 04 Hetero chromosome compartment 12 04 Chromosome Compartments Frequency Hetero 12 Hetero 04 1459 0.367 0.214 10 91 0.418 0,066 twenty 135 0.311 0,081 thirty 185 0.384 0.2 40 169 0.355 0.314 fifty 157 0.357 0.35 60 153 0.346 0.209 70 123 0,358 0.163 80 113 0.292 0.195 90 118 0.398 0.254 one hundred 88 0.477 0.273 110 49 0.388 0.224 120 29th 0.241 0.172 130 26 0.385 0.192 140 twenty 0.6 0.05 150 3 0.667 0 one 142 0.366 0.324 2 150 0.3 0.193 3 111 0.459 0.261 four 133 0,301 0.211 5 121 0.388 0.273 6 115 0.443 0.217 7 103 0.369 0.136 8 93 0.43 0.172 9 66 0.273 0.167 10 88 0.352 0.284 eleven 78 0.372 0.154 12 77 0.39 0.143 13 78 0.346 0.167 fourteen 62 0.403 0.274 fifteen 42 0.286 0,071 one twenty 9 0.333 0,111 one thirty fourteen 0.214 0.214 one 40 12 0.333 0.333 one fifty fifteen 0.4 0.4 one 60 21 0.381 0.381 one 70 19 0.421 0.421 one 80 9 0.333 0.333 one 90 13 0.385 0.385 one one hundred 13 0,308 0.231 one 110 8 0.5 0.25 one 120 6 0.167 0 one 130 3 one one 2 10 3 0,333J 0 2 twenty eleven 0.364 0.182 2 thirty 13 0.231 0.231 2 40 6 0.333 0.333 2 fifty 19 0.421 0.421 2 60 13 0.385 0.385 2 70 10 0.2 0.2 2 80 13 0.154 0.154 2 90 19 0.158 0.158 2 one hundred 7 0 0 2 110 four 0.25 0.25 2 120 7 0.286 0.143 2 130 eleven 0.364 0 2 140 eleven 0.545 0 2 150 3 0.667 0 3 10 9 0.444 0 3 twenty fifteen 0.467 0.133 3 thirty 12 0.5 0.25 3 40 eleven 0.182 0 3 fifty 3 0 0 3 60 12 0.583 0.333 3 70 12 0.5 0,083 3 80 fourteen 0.429 0.429 3 90 12 0.5 0.5 3 one hundred 9 0.667 0.667 3 110 2 0.5 0.5 four 10 13 0.385 0.231 four twenty 9 0.333 0.222 four thirty fifteen 0.267 0.133 four 40 fifteen 0.333 0.267 four fifty eleven 0.455 0.455 four 60 8 0.25 0.25 four 70 7 0 0 four 80 9 0.333 0.333 four 90 9 0.333 0.333 four one hundred 7 0.286 0.286 four 110 8 0.25 0.25 four 120 7 0 0 four 130 8 0.125 0 four 140 7 0.714 0 5 10 12 0.5 0.167 5 twenty 12 0.333 0.167 5 thirty eleven 0.273 0.273 5 40 fourteen 0.429 0.429 5 fifty 10 0.1 0.1 5 60 8 0.25 0.25 5 70 8 0.375 0.375 5 80 8 0.375 0.25 5 90 8 0.25 0 5 one hundred 7 0.714 0 5 110 9 0.556 0.556 5 120 8 0.5 0.5 5 130 four 0.5 0.5 5 140 2 0.5 0.5 6 10 13 0.385 0 6 twenty fourteen 0.214 0 6 thirty eleven 0.455 0.364 6 40 12 0.75 0.75 6 fifty 10 0.5 0.5 6 60 eighteen 0.389 0,111 6 70 8 0.5 0 6 80 8 0.125 0 6 90 7 0.429 0.429 6 one hundred 10 0.7 0.2 6 110 four 0.5 0 7 10 6 0.333 0 7 twenty 12 0.5 0 7 thirty 16 0.25 0 7 40 fourteen 0.357 0.357 7 fifty 16 0.438 0.438 7 60 fifteen 0.267 0 7 70 9 0.333 0 7 80 5 0.4 0 7 90 7 0.429 0 7 one hundred 3 0.667 0.667 8 10 8 0.25 0 8 twenty 10 0.2 0 8 thirty fifteen 0.667 0 8 40 13 0.385 0.385 8 fifty eleven 0.273 0.273 8 60 four 0.25 0.25 8 70 10 0.5 0.5 8 80 6 0.333 0.167 8 90 eleven 0.636 0 8 one hundred 5 0.6 0.2 9 twenty 9 0,111 0 9 thirty fifteen 0.4 0.2 9 40 10 0.1 0.1 9 fifty 13 0.385 0.385 9 60 9 0.222 0.222 9 70 6 0.333 0 9 80 four 0.25 0 10 10 7 0.571 0.143 10 twenty fifteen 0.267 0.133 10 thirty 19 0.368 0.368 10 40 12 0.167 0.167 10 fifty 6 0.333 0.333 10 60 9 0.333 0.333 10 70 2 0.5 0.5 10 80 8 0.375 0.375 10 90 7 0.571 0.571 10 one hundred 3 0.333 0 eleven 10 7 0.286 0 eleven twenty 8 0.375 0 eleven thirty fifteen 0.4 0.2 eleven 40 fifteen 0.467 0.2 eleven fifty 16 0.313 0.313 eleven 60 7 0.143 0.143 eleven 70 6 0.5 0 eleven 80 four 0.5 0 12 10 13 0.538 0 12 twenty 9 0.222 0 12 thirty 13 0.462 0,077 12 40 12 0.333 0.333 12 fifty 9 0.444 0.444 12 60 5 0.2 0.2 12 70 7 0 0 12 80 3 0.333 0.333 12 90 four 0.75 0 12 one hundred 2 one 0 13 twenty one 0 0 13 thirty four 0.5 0.5 13 40 10 0.2 0.2 13 fifty 7 0.286 0.143 13 60 9 0.444 0 13 70 8 0.5 0 13 80 9 0,111 0,111 13 90 7 0.429 0.429 13 one hundred eleven 0.455 0.364 13 110 eleven 0.364 0 13 120 one 0 0 fourteen twenty one 0 0 fourteen thirty 7 0.714 0.429 fourteen 40 8 0.625 0.625 fourteen fifty 8 0.375 0.375 fourteen 60 9 0.333 0,111 fourteen 70 6 0.333 0 fourteen 80 8 0.125 0 fourteen 90 7 0.286 0.286 fourteen one hundred 6 0.667 0.5 fourteen 110 2 0 0 fifteen thirty 5 0.2 0 fifteen 40 5 0.2 0.2 fifteen fifty 3 0 0 fifteen 60 6 0.5 0 fifteen 70 5 0.2 0 fifteen 80 5 0.4 0 fifteen 90 7 0.429 0.143 fifteen one hundred 5 0.2 0.2 fifteen 110 one 0 0

Table E
Chromosome compartments, heterozygosity / frequency on chromosomes 12 and 05 Hetero chromosome compartment 12 05 Chromosome Compartments Frequency Hetero 12 Hetero 05 1459 0.367 0.214 10 91 0.418 0,066 twenty 135 0.311 0,081 thirty 185 0.384 0.2 40 169 0.355 0.314 fifty 157 0.357 0.35 60 153 0.346 0.209 70 123 0,358 0.163 80 113 0.292 0.195 90 118 0.398 0.254 one hundred 88 0.477 0.273 110 49 0.388 0.224 120 29th 0.241 0.172 130 26 0.385 0.192 140 twenty 0.6 0.05 150 3 0.667 0 one 142 0.366 0.324 2 150 0.3 0.193 3 111 0.459 0.261 four 133 0,301 0.211 5 121 0.388 0.273 6 115 0.443 0.217 7 103 0.369 0.136 8 93 0.43 0.172 9 66 0.273 0.167 10 88 0.352 0.284 eleven 78 0.372 0.154 12 77 0.39 0.143 13 78 0.346 0.167 fourteen 62 0.403 0.274 fifteen 42 0.286 0,071 one twenty 9 0.333 0,111 one thirty fourteen 0.214 0.214 one 40 12 0.333 0.333 one fifty fifteen 0.4 0.4 one 60 21 0.381 0.381 one 70 19 0.421 0.421 one 80 9 0.333 0.333 one 90 13 0.385 0.385 one one hundred 13 0,308 0.231 one 110 8 0.5 0.25 one 120 6 0.167 0 one 130 3 one one 2 10 3 0.333 0 2 twenty eleven 0.364 0.182 2 thirty 13 0.231 0.231 2 40 6 0.333 0.333 2 fifty 19 0.421 0.421 2 60 13 0.385 0.385 2 70 10 0.2 0.2 2 80 13 0.154 0.154 2 90 19 0.158 0.158 2 one hundred 7 0 0 2 110 four 0.25 0.25 2 120 7 0.286 0.143 2 130 eleven 0.364 0 2 140 eleven 0.545 0 2 150 3 0.667 0 3 10 9 0.444 0 3 twenty fifteen 0.467 0.133 3 thirty 12 0.5 0.25 3 40 eleven 0.182 0 3 fifty 3 0 0 3 60 12 0.583 0.333 3 70 12 0.5 0,083 3 80 fourteen 0.429 0.429 3 90 12 0.5 0.5 3 one hundred 9 0.667 0.667 3 110 2 0.5 0.5 four 10 13 0.385 0.231 four twenty 9 0.333 0.222 four thirty fifteen 0.267 0.133 four 40 fifteen 0.333 0.267 four fifty eleven 0.455 0.455 four 60 8 0.25 0.25 four 70 7 0 0 four 80 9 0.333 0.333 four 90 9 0.333 0.333 four one hundred 7 0.286 0.286 four 110 8 0.25 0.25 four 120 7 0 0 four 130 8 0.125 0 four 140 7 0.714 0 5 10 12 0.5 0.167 5 twenty 12 0.333 0.167 5 thirty eleven 0.273 0.273 5 40 fourteen 0.429 0.429 5 fifty 10 0.1 0.1 5 60 8 0.25 0.25 5 70 8 0.375 0.375 5 80 8 0.375 0.25 5 90 8 0.25 0 5 one hundred 7 0.714 0 5 110 9 0.556 0.556 5 120 8 0.5 0.5 5 130 four 0.5 0.5 5 140 2 0.5 0.5 6 10 13 0.385 0 6 twenty fourteen 0.214 0 6 thirty eleven 0.455 0.364 6 40 12 0.75 0.75 6 fifty 10 0.5 0.5 6 60 eighteen 0.389 0,111 6 70 8 0.5 0 6 80 8 0.125 0 6 90 7 0.429 0.429 6 one hundred 10 0.7 0.2 6 110 four 0.5 0 7 10 6 0.333 0 7 twenty 12 0.5 0 7 thirty 16 0.25 0 7 40 fourteen 0.357 0.357 7 fifty 16 0.438 0.438 7 60 fifteen 0.267 0 7 70 9 0.333 0 7 80 5 0.4 0 7 90 7 0.429 0 7 one hundred 3 0.667 0.667 8 10 8 0.25 0 8 twenty 10 0.2 0 8 thirty fifteen 0.667 0 8 40 13 0.385 0.385 8 fifty eleven 0.273 0.273 8 60 four 0.25 0.25 8 70 10 0.5 0.5 8 80 6 0.333 0.167 8 90 eleven 0.636 0 8 one hundred 5 0.6 0.2 9 twenty 9 0,111 0 9 thirty fifteen 0.4 0.2 9 40 10 0.1 0.1 9 fifty 13 0.385 0.385 9 60 9 0.222 0.222 9 70 6 0.333 0 9 80 four 0.25 0 10 10 7 0.571 0.143 10 twenty fifteen 0.267 0.133 10 thirty 19 0.368 0.368 10 40 12 0.167 0.167 10 fifty 6 0.333 0.333 10 60 9 0.333 0.333 10 70 2 0.5 0.5 10 80 8 0.375 0.375 10 90 7 0.571 0.571 10 one hundred 3 0.333 0 eleven 10 7 0.286 0 eleven twenty 8 0.375 0 eleven thirty fifteen 0.4 0.2 eleven 40 fifteen 0.467 0.2 eleven fifty 16 0.313 0.313 eleven 60 7 0.143 0.143 eleven 70 6 0.5 0 eleven 80 four 0.5 0 12 10 13 0.538 0 12 twenty 9 0.222 0 12 thirty 13 0.462 0,077 12 40 12 0.333 0.333 12 fifty 9 0.444 0.444 12 60 5 0.2 0.2 12 70 7 0 0 12 80 3 0.333 0.333 12 90 four 0.75 0 12 one hundred 2 one 0 13 twenty one 0 0 13 thirty four 0.5 0.5 13 40 10 0.2 0.2 13 fifty 7 0.286 0.143 13 60 9 0.444 0 13 70 8 0.5 0 13 80 9 0,111 0,111 13 90 7 0.429 0.429 13 one hundred eleven 0.455 0.364 13 110 eleven 0.364 0 13 120 one 0 0 fourteen twenty one 0 0 fourteen thirty 7 0.714 0.429 fourteen 40 8 0.625 0.625 fourteen fifty 8 0.375 0.375 fourteen 60 9 0.333 0,111 fourteen 70 6 0.333 0 fourteen 80 8 0.125 0 fourteen 90 7 0.286 0.286 fourteen one hundred 6 0.667 0.5 fourteen 110 2 0 0 fifteen thirty 5 0.2 0 fifteen 40 5 0.2 0.2 fifteen fifty 3 0 0 fifteen 60 6 0.5 0 fifteen 70 5 0.2 0 fifteen 80 5 0.4 0 fifteen 90 7 0.429 0.143 fifteen one hundred 5 0.2 0.2 fifteen 110 one 0 0

Table F
Chromosomal compartments, heterozygosity / frequency for chromosomes 11 and 16 Hetero chromosome compartment 11 06 Chromosome Compartments Frequency Hetero 11 Hetero 06 1459 0.326 0.213 10 91 0.33 0,077 twenty 135 0.319 0,096 thirty 185 0.346 0.2 40 169 0.367 0.243 fifty 157 0.344 0.223 60 153 0.294 0.222 70 123 0.276 0.252 80 113 0.345 0.292 90 118 0.254 0.212 one hundred 88 0.33 0.193 110 49 0.327 0.245 120 29th 0.345 0.345 130 26 0.423 0.346 140 twenty 0.4 0.35 150 3 0 0 one 142 0.317 0.197 2 150 0.273 0,207 3 111 0.288 0.234 four 133 0.338 0.173 5 121 0.355 0.331 6 115 0.252 0,096 7 103 0.34 0.204 8 93 0.344 0.204 9 66 0.318 0.167 10 88 0.432 0,307 eleven 78 0.372 0.167 12 77 0.39 0.169 13 78 0.244 0.154 fourteen 62 0.323 0.323 fifteen 42 0.381 0.381 one twenty 9 0.333 0 one thirty fourteen 0.429 0.286 one 40 12 0.333 0.167 one fifty fifteen 0.333 0.267 one 60 21 0.286 0.286 one 70 19 0.211 0.211 one 80 9 0.222 0,111 one 90 13 0.231 0 one one hundred 13 0.462 0,308 one 110 8 0.25 0.125 one 120 6 0.333 0.333 one 130 3 0.667 0 2 10 3 0 0 2 twenty eleven 0.455 0 2 thirty 13 0,308 0 2 40 6 0 0 2 fifty 19 0.211 0.211 2 60 13 0.462 0.462 2 70 10 0.2 0.2 2 80 13 0.231 0.231 2 90 19 0.368 0.316 2 one hundred 7 0 0 2 110 four 0.25 0.25 2 120 7 0.143 0.143 2 130 eleven 0.364 0.364 2 140 eleven 0.364 0.364 2 150 3 0 0 3 10 9 0.333 0 3 twenty fifteen 0,067 0 3 thirty 12 0.25 0,083 3 40 eleven 0.545 0.545 3 fifty 3 0.333 0.333 3 60 12 0.167 0.167 3 70 12 0.333 0.333 3 80 fourteen 0.5 0.5 3 90 12 0.167 0.167 3 one hundred 9 0.333 0.333 3 110 2 0 0 four 10 13 0,308 0.154 four twenty 9 0.222 0,111 four thirty fifteen 0.333 0.267 four 40 fifteen 0.467 0.2 four fifty eleven 0.273 0 four 60 8 0.5 0 four 70 7 0.286 0 four 80 9 0.333 0 four 90 9 0,111 0 four one hundred 7 0.286 0.286 four 110 8 0.25 0.25 four 120 7 0.429 0.429 four 130 8 0.5 0.5 four 140 7 0.429 0.286 5 10 12 0.333 0,083 5 twenty 12 0,083 0,083 5 thirty eleven 0.455 0.455 5 40 fourteen 0.286 0.286 5 fifty 10 0.5 0.5 5 60 8 0.125 0.125 5 70 8 0.375 0.375 5 80 8 0.375 0.375 5 90 8 0.125 0.125 5 one hundred 7 0.571 0.571 5 110 9 0.667 0.667 5 120 8 0.5 0.5 5 130 four 0.25 0.25 5 140 2 0.5 0.5 6 10 13 0.231 0 6 twenty fourteen 0.143 0.143 6 thirty eleven 0.182 0,091 6 40 12 0.333 0.167 6 fifty 10 0.3 0.1 6 60 eighteen 0.333 0.056 6 70 8 0.125 0.125 6 80 8 0.25 0.25 6 90 7 0.143 0.143 6 one hundred 10 0.4 0 6 110 four 0.25 0 7 10 6 0.167 0 7 twenty 12 0.25 0 7 thirty 16 0.25 0,063 7 40 fourteen 0.357 0.286 7 fifty 16 0.563 0.313 7 60 fifteen 0.333 0.2 7 70 9 0.444 0.444 7 80 5 0.4 0.4 7 90 7 0.143 0.143 7 one hundred 3 0.333 0.333 8 10 8 0.5 0 8 twenty 10 0.7 0.1 8 thirty fifteen 0.333 0.333 8 40 13 0.231 0.231 8 fifty eleven 0.182 0.182 8 60 four 0 0 8 70 10 0.4 0.4 8 80 6 0.167 0.167 8 90 eleven 0.273 0.273 8 one hundred 5 0.6 0 9 twenty 9 0.222 0 9 thirty fifteen 0.4 0,067 9 40 10 0.6 0.3 9 fifty 13 0,077 0,077 9 60 9 0.333 0.333 9 70 6 0.333 0.333 9 80 four 0.25 0.25 10 10 7 0.571 0.571 10 twenty fifteen 0.6 0.267 10 thirty 19 0.368 0.316 10 40 12 0.417 0.25 10 fifty 6 0.833 0.333 10 60 9 0.444 0.444 10 70 2 0 0 10 80 8 0.375 0.375 10 90 7 0 0 10 one hundred 3 0.333 0.333 eleven 10 7 0.429 0 eleven twenty 8 0.625 0.125 eleven thirty fifteen 0.467 0.267 eleven 40 fifteen 0.267 0.2 eleven fifty 16 0.375 0.25 eleven 60 7 0 0 eleven 70 6 0.333 0.167 eleven 80 four 0.5 0 12 10 13 0,308 0 12 twenty 9 0.333 0.333 12 thirty 13 0.462 0.154 12 40 12 0.583 0,083 12 fifty 9 0.333 0 12 60 5 0.2 0.2 12 70 7 0.286 0.286 12 80 3 0.333 0.333 12 90 four 0.75 0.75 12 one hundred 2 0 0 13 twenty one 0 0 13 thirty four 0.25 0 13 40 10 0 0 13 fifty 7 0.429 0.286 13 60 9 0,111 0,111 13 70 8 0.25 0.25 13 80 9 0.444 0.444 13 90 7 0.429 0.429 13 one hundred eleven 0.273 0 13 110 eleven 0.182 0 13 120 one 0 0 fourteen twenty one 0 0 fourteen thirty 7 0.143 0.143 fourteen 40 8 0.625 0.625 fourteen fifty 8 0.375 0.375 fourteen 60 9 0.444 0.444 fourteen 70 6 0.167 0.167 fourteen 80 8 0.25 0.25 fourteen 90 7 0.143 0.143 fourteen one hundred 6 0.167 0.167 fourteen 110 2 one one fifteen thirty 5 0.4 0.4 fifteen 40 5 0.4 0.4 fifteen fifty 3 0.333 0.333 fifteen 60 6 0.333 0.333 fifteen 70 5 0.2 0.2 fifteen 80 5 0.6 0.6 fifteen 90 7 0.571 0.571 fifteen one hundred 5 0.2 0.2 fifteen 110 one 0 0

Table g
Chromosome compartments, heterozygosity / frequency for chromosomes 11 and 07 Hetero chromosome compartment 11 07 Chromosome Compartments Frequency Hetero 11 Hetero 07 1459 0.326 0.168 10 91 0.33 0,033 twenty 135 0.319 0,081 thirty 185 0.346 0,092 40 169 0.367 0.16 fifty 157 0.344 0.185 60 153 0.294 0.176 70 123 0.276 0.154 80 113 0.345 0.274 90 118 0.254 0.186 one hundred 88 0.33 0.239 110 49 0.327 0.265 120 29th 0.345 0.276 130 26 0.423 0.346 140 twenty 0.4 0.4 150 3 0 0 one 142 0.317 0,085 2 150 0.273 0,093 3 111 0.288 0.189 four 133 0.338 0.12 5 121 0.355 0.215 6 115 0.252 0.217 7 103 0.34 0.282 8 93 0.344 0,097 9 66 0.318 0,061 10 88 0.432 0.239 eleven 78 0.372 0.218 12 77 0.39 0,091 13 78 0.244 0.154 fourteen 62 0.323 0.258 fifteen 42 0.381 0.381 one twenty 9 0.333 0 one thirty fourteen 0.429 0 one 40 12 0.333 0 one fifty fifteen 0.333 0 one 60 21 0.286 0 one 70 19 0.211 0,053 one 80 9 0.222 0,111 one 90 13 0.231 0.231 one one hundred 13 0.462 0.154 one 110 8 0.25 0.25 one 120 6 0.333 0.167 one 130 3 0.667 0.667 2 10 3 0 0 2 twenty eleven 0.455 0 2 thirty 13 0,308 0 2 40 6 0 0 2 fifty 19 0.211 0 2 60 13 0.462 0.231 2 70 10 0.2 0 2 80 13 0.231 0.154 2 90 19 0.368 0.158 2 one hundred 7 0 0 2 110 four 0.25 0 2 120 7 0.143 0 2 130 eleven 0.364 0.182 2 140 eleven 0.364 0.364 2 150 3 0 0 3 10 9 0.333 0,111 3 twenty fifteen 0,067 0 3 thirty 12 0.25 0,083 3 40 eleven 0.545 0.364 3 fifty 3 0.333 0.333 3 60 12 0.167 0.167 3 70 12 0.333 0.333 3 80 fourteen 0.5 0.357 3 90 12 0.167 0 3 one hundred 9 0.333 0.333 3 110 2 0 0 four 10 13 0,308 0 four twenty 9 0.222 0 four thirty fifteen 0.333 0 four 40 fifteen 0.467 0,067 four fifty eleven 0.273 0 four 60 8 0.5 0 four 70 7 0.286 0 four 80 9 0.333 0 four 90 9 0,111 0,111 four one hundred 7 0.286 0.286 four 110 8 0.25 0.25 four 120 7 0.429 0.429 four 130 8 0.5 0.5 four 140 7 0.429 0.429 5 10 12 0.333 0 5 twenty 12 0,083 0 5 thirty eleven 0.455 0 5 40 fourteen 0.286 0,071 5 fifty 10 0.5 0.3 5 60 8 0.125 0 5 70 8 0.375 0.25 5 80 8 0.375 0.375 5 90 8 0.125 0.125 5 one hundred 7 0.571 0.571 5 110 9 0.667 0.667 5 120 8 0.5 0.5 5 130 four 0.25 0.25 5 140 2 0.5 0.5 6 10 13 0.231 0 6 twenty fourteen 0.143 0.143 6 thirty eleven 0.182 0,091 6 40 12 0.333 0.333 6 fifty 10 0.3 0.3 6 60 eighteen 0.333 0.333 6 70 8 0.125 0.125 6 80 8 0.25 0.25 6 90 7 0.143 0.143 6 one hundred 10 0.4 0.4 6 110 four 0.25 0.25 7 10 6 0.167 0 7 twenty 12 0.25 0,083 7 thirty 16 0.25 0.125 7 40 fourteen 0.357 0.286 7 fifty 16 0.563 0.563 7 60 fifteen 0.333 0.333 7 70 9 0.444 0.444 7 80 5 0.4 0.4 7 90 7 0.143 0.143 7 one hundred 3 0.333 0.333 8 10 8 0.5 0 8 twenty 10 0.7 0 8 thirty fifteen 0.333 0 8 40 13 0.231 0.154 8 fifty eleven 0.182 0.182 8 60 four 0 0 8 70 10 0.4 0 8 80 6 0.167 0 8 90 eleven 0.273 0.182 8 one hundred 5 0.6 0.6 9 twenty 9 0.222 0 9 thirty fifteen 0.4 0 9 40 10 0.6 0 9 fifty 13 0,077 0,077 9 60 9 0.333 0,111 9 70 6 0.333 0.167 9 80 four 0.25 0.25 10 10 7 0.571 0.286 10 twenty fifteen 0.6 0.267 10 thirty 19 0.368 0.316 10 40 12 0.417 0.25 10 fifty 6 0.833 0 10 60 9 0.444 0.333 10 70 2 0 0 10 80 8 0.375 0.375 10 90 7 0 0 10 one hundred 3 0.333 0 eleven 10 7 0.429 0 eleven twenty 8 0.625 0.5 eleven thirty fifteen 0.467 0.2 eleven 40 fifteen 0.267 0.2 eleven fifty 16 0.375 0.188 eleven 60 7 0 0 eleven 70 6 0.333 0.333 eleven 80 four 0.5 0.5 12 10 13 0,308 0 12 twenty 9 0.333 0 12 thirty 13 0.462 0.154 12 40 12 0.583 0,083 12 fifty 9 0.333 0 12 60 5 0.2 0 12 70 7 0.286 0 12 80 3 0.333 0.333 12 90 four 0.75 0.75 12 one hundred 2 0 0 13 twenty one 0 0 13 thirty four 0.25 0 13 40 10 0 0 13 fifty 7 0.429 0.429 13 60 9 0,111 0,111 13 70 8 0.25 0.25 13 80 9 0.444 0.444 13 90 7 0.429 0.286 13 one hundred eleven 0.273 0 13 110 eleven 0.182 0 13 120 one 0 0 fourteen twenty one 0 0 fourteen thirty 7 0.143 0 fourteen 40 8 0.625 0.25 fourteen fifty 8 0.375 0.375 fourteen 60 9 0.444 0.444 fourteen 70 6 0.167 0.167 fourteen 80 8 0.25 0.25 fourteen 90 7 0.143 0.143 fourteen one hundred 6 0.167 0.167 fourteen 110 2 one one fifteen thirty 5 0.4 0.4 fifteen 40 5 0.4 0.4 fifteen fifty 3 0.333 0.333 fifteen 60 6 0.333 0.333 fifteen 70 5 0.2 0.2 fifteen 80 5 0.6 0.6 fifteen 90 7 0.571 0.571 fifteen one hundred 5 0.2 0.2 fifteen 110 one 0 0

Table h
Chromosome compartments, heterozygosity / frequency for chromosomes 11 and 08 Hetero chromosome compartment 11 08 Chromosome Compartments Frequency Hetero 11 Hetero 08 1459 0.326 0.206 10 91 0.33 0,066 twenty 135 0.319 0.163 thirty 185 0.346 0.27 40 169 0.367 0.302 fifty 157 0.344 0.21 60 153 0.294 0.157 70 123 0.276 0.187 80 113 0.345 0.195 90 118 0.254 0.136 one hundred 88 0.33 0.25 110 49 0.327 0.265 120 29th 0.345 0.241 130 26 0.423 0.231 140 twenty 0.4 0.25 150 3 0 0 one 142 0.317 0.169 2 150 0.273 0.187 3 111 0.288 0.153 four 133 0.338 0.15 5 121 0.355 0.248 6 115 0.252 0.165 7 103 0.34 0.282 8 93 0.344 0.269 9 66 0.318 0.288 10 88 0.432 0.227 eleven 78 0.372 0.256 12 77 0.39 0.169 13 78 0.244 0.231 fourteen 62 0.323 0.145 fifteen 42 0.381 0.214 one twenty 9 0.333 0 one thirty fourteen 0.429 0.286 one 40 12 0.333 0.333 one fifty fifteen 0.333 0.333 one 60 21 0.286 0.143 one 70 19 0.211 0.105 one 80 9 0.222 0 one 90 13 0.231 0 one one hundred 13 0.462 0,077 one 110 8 0.25 0.125 one 120 6 0.333 0.333 one 130 3 0.667 0.667 2 10 3 0 0 2 twenty eleven 0.455 0.273 2 thirty 13 0,308 0,308

2 40 6 0 0 2 fifty 19 0.211 0.211 2 60 13 0.462 0.231 2 70 10 0.2 0 2 80 13 0.231 0,077 2 90 19 0.368 0.158 2 one hundred 7 0 0 2 110 four 0.25 0.25 2 120 7 0.143 0.143 2 130 eleven 0.364 0.364 2 140 eleven 0.364 0.364 2 150 3 0 0 3 10 9 0.333 0 3 twenty fifteen 0,067 0,067 3 thirty 12 0.25 0.25 3 40 eleven 0.545 0.545 3 fifty 3 0.333 0.333 3 60 12 0.167 0.167 3 70 12 0.333 0,083 3 80 fourteen 0.5 0 3 90 12 0.167 0 3 one hundred 9 0.333 0.333 3 110 2 0 0 four 10 13 0,308 0,077 four twenty 9 0.222 0.222 four thirty fifteen 0.333 0.333 four 40 fifteen 0.467 0.2 four fifty eleven 0.273 0 four 60 8 0.5 0 four 70 7 0.286 0.286 four 80 9 0.333 0.333 four 90 9 0,111 0,111 four one hundred 7 0.286 0.286 four 110 8 0.25 0 four 120 7 0.429 0 four 130 8 0.5 0 four 140 7 0.429 0.143 5 10 12 0.333 0.167 5 twenty 12 0,083 0,083 5 thirty eleven 0.455 0.455 5 40 fourteen 0.286 0.286 5 fifty 10 0.5 0.1 5 60 8 0.125 0 5 70 8 0.375 0 5 80 8 0.375 0.25 5 90 8 0.125 0.125 5 one hundred 7 0.571 0.571 5 110 9 0.667 0.667 5 120 8 0.5 0.5 5 130 four 0.25 0 5 140 2 0.5 0 6 10 13 0.231 0 6 twenty fourteen 0.143 0.143 6 thirty eleven 0.182 0.182 6 40 12 0.333 0.333 6 fifty 10 0.3 0.1 6 60 eighteen 0.333 0.056 6 70 8 0.125 0.125 6 80 8 0.25 0.25 6 90 7 0.143 0.143 6 one hundred 10 0.4 0.4 6 110 four 0.25 0.25 7 10 6 0.167 0 7 twenty 12 0.25 0.167 7 thirty 16 0.25 0.25 7 40 fourteen 0.357 0.357 7 fifty 16 0.563 0.375 7 60 fifteen 0.333 0.333 7 70 9 0.444 0.444 7 80 5 0.4 0.4 7 90 7 0.143 0.143 7 one hundred 3 0.333 0 8 10 8 0.5 0.25 8 twenty 10 0.7 0.3 8 thirty fifteen 0.333 0.333 8 40 13 0.231 0.231 8 fifty eleven 0.182 0,091 8 60 four 0 0 8 70 10 0.4 0.4 8 80 6 0.167 0.167 8 90 eleven 0.273 0.273 8 one hundred 5 0.6 0.6 9 twenty 9 0.222 0 9 thirty fifteen 0.4 0.4 9 40 10 0.6 0.6 9 fifty 13 0,077 0,077 9 60 9 0.333 0.333 9 70 6 0.333 0.333 9 80 four 0.25 0.25 10 10 7 0.571 0 10 twenty fifteen 0.6 0.133 10 thirty 19 0.368 0,053 10 40 12 0.417 0.417 10 fifty 6 0.833 0.833 10 60 9 0.444 0.444 10 70 2 0 0 10 80 8 0.375 0.375 10 90 7 0 0 10 one hundred 3 0.333 0 eleven 10 7 0.429 0 eleven twenty 8 0.625 0.375 eleven thirty fifteen 0.467 0.467 eleven 40 fifteen 0.267 0.2 eleven fifty 16 0.375 0.188 eleven 60 7 0 0 eleven 70 6 0.333 0.333 eleven 80 four 0.5 0.5 12 10 13 0,308 0,077 12 twenty 9 0.333 0.333 12 thirty 13 0.462 0.154 12 40 12 0.583 0,083 12 fifty 9 0.333 0 12 60 5 0.2 0 12 70 7 0.286 0.286 12 80 3 0.333 0.333 12 90 four 0.75 0.75 12 one hundred 2 0 0 13 twenty one 0 0 13 thirty four 0.25 0 13 40 10 0 0 13 fifty 7 0.429 0.429 13 60 9 0,111 0,111 13 70 8 0.25 0.25 13 80 9 0.444 0.444 13 90 7 0.429 0.429 13 one hundred eleven 0.273 0.273 13 110 eleven 0.182 0.182 13 120 one 0 0 fourteen twenty one 0 0 fourteen thirty 7 0.143 0 fourteen 40 8 0.625 0.625 fourteen fifty 8 0.375 0.125 fourteen 60 9 0.444 0 fourteen 70 6 0.167 0 fourteen 80 8 0.25 0 fourteen 90 7 0.143 0 fourteen one hundred 6 0.167 0.167 fourteen 110 2 one one fifteen thirty 5 0.4 0.4 fifteen 40 5 0.4 0.4 fifteen fifty 3 0.333 0.333 fifteen 60 6 0.333 0.333 fifteen 70 5 0.2 0.2 fifteen 80 5 0.6 0 fifteen 90 7 0.571 0 fifteen one hundred 5 0.2 0.2 fifteen 110 one 0 0

Table I
Chromosome compartments, heterozygosity / frequency for chromosomes 10 and 09 Hetero chromosome compartment 10 09 Chromosome Compartments Frequency Hetero 10 Hetero 09 1459 0.378 0.222 10 91 0.473 0,066 twenty 135 0.319 0.178 thirty 185 0.314 0.227 40 169 0.467 0.402 fifty 157 0.357 0.21 60 153 0.405 0.216 70 123 0,358 0.138 80 113 0.363 0.257 90 118 0.373 0.237 one hundred 88 0.432 0.216 110 49 0.408 0.184 120 29th 0.31 0,207 130 26 0,308 0.269 140 twenty 0.2 0.1 150 3 one 0.333 one 142 0.387 0.218 2 150 0.353 0.253 3 111 0.405 0.243 four 133 0.346 0.218 5 121 0.413 0.198 6 115 0.409 0.243 7 103 0.359 0.233 8 93 0.398 0.118 9 66 0.333 0.273 10 88 0.341 0.273 eleven 78 0.372 0.167 12 77 0.429 0.182 13 78 0.423 0.256 fourteen 62 0.339 0.226 fifteen 42 0.333 0.214 one twenty 9 0.556 0.222 one thirty fourteen 0.357 0,071 one 40 12 0.333 0.333 one fifty fifteen 0.267 0.2 one 60 21 0.429 0.286 one 70 19 0.526 0.263 one 80 9 0.222 0.222 one 90 13 0,077 0 one one hundred 13 0.385 0,308 one 110 8 0.625 0.125 one 120 6 0.5 0.167 one 130 3 0.667 0.667 2 10 3 0.333 0 2 twenty eleven 0.364 0.364 2 thirty 13 0,308 0,308 2 40 6 0 0 2 fifty 19 0.263 0.263 2 60 13 0.615 0.231 2 70 10 0.3 0.2 2 80 13 0.538 0.385 2 90 19 0.421 0.368 2 one hundred 7 0.571 0.571 2 110 four 0 0 2 120 7 0.286 0.286 2 130 eleven 0.182 0,091 2 140 eleven 0.182 0 2 150 3 one 0.333 3 10 9 0.333 0 3 twenty fifteen 0.4 0,067 3 thirty 12 0.333 0.25 3 40 eleven 0.636 0.636 3 fifty 3 0 0 3 60 12 0.333 0.25 3 70 12 0.417 0.25 3 80 fourteen 0.571 0.357 3 90 12 0.25 0.167 3 one hundred 9 0.444 0.222 3 110 2 0.5 0.5 four 10 13 0.615 0.154 four twenty 9 0,111 0,111 four thirty fifteen 0.2 0,067 four 40 fifteen 0.267 0.133 four fifty eleven 0.545 0.182 four 60 8 0.375 0.375 four 70 7 0 0 four 80 9 0.222 0.222 four 90 9 0.667 0.667 four one hundred 7 0.286 0 four 110 8 0.5 0.375 four 120 7 0.429 0.429 four 130 8 0.25 0.25 four 140 7 0.286 0.286 5 10 12 0.417 0 5 twenty 12 0.25 0.167 5 thirty eleven 0.455 0.455 5 40 fourteen 0.5 0.357 5 fifty 10 0.7 0.2 5 60 8 0.375 0 5 70 8 0.25 0 5 80 8 0.375 0.375 5 90 8 0.5 0.5 5 one hundred 7 0.571 0 5 110 9 0.444 0,111 5 120 8 0.125 0 5 130 four 0.5 0.5 5 140 2 0 0 6 10 13 0.462 0.154 6 twenty fourteen 0.286 0.143 6 thirty eleven 0.364 0.364 6 40 12 0.583 0.583 6 fifty 10 0.3 0.3 6 60 eighteen 0.278 0.278 6 70 8 0.375 0.375 6 80 8 0.375 0.25 6 90 7 0.714 0 6 one hundred 10 0.7 0 6 110 four 0 0 7 10 6 0.5 0.167 7 twenty 12 0.25 0.25 7 thirty 16 0.313 0.313 7 40 fourteen 0.571 0.571 7 fifty 16 0.25 0.25 7 60 fifteen 0.6 0.2 7 70 9 0.444 0 7 80 5 0.2 0 7 90 7 0 0 7 one hundred 3 0 0 8 10 8 0.25 0 8 twenty 10 0.4 0.3 8 thirty fifteen 0.467 0.2 8 40 13 0,308 0.154 8 fifty eleven 0.364 0.182 8 60 four 0.5 0.25 8 70 10 0.5 0 8 80 6 0.5 0 8 90 eleven 0.455 0 8 one hundred 5 0.2 0 9 twenty 9 0.333 0,111 9 thirty fifteen 0.133 0,067 9 40 10 0.6 0.5 9 fifty 13 0.385 0.385 9 60 9 0.333 0.333 9 70 6 0.167 0.167 9 80 four 0.5 0.5 10 10 7 0.571 0 10 twenty fifteen 0.267 0.133 10 thirty 19 0.263 0.263 10 40 12 0.417 0.417 10 fifty 6 0.167 0.167 10 60 9 0.222 0.222 10 70 2 0.5 0.5 10 80 8 0.375 0.375 10 90 7 0.571 0.571 10 one hundred 3 0.333 0.333 eleven 10 7 0.571 0.143 eleven twenty 8 0.25 0 eleven thirty fifteen 0.333 0.333 eleven 40 fifteen 0.267 0.267 eleven fifty 16 0.563 0.188 eleven 60 7 0.286 0 eleven 70 6 0.5 0 eleven 80 four 0 0 12 10 13 0.538 0 12 twenty 9 0.333 0.333 12 thirty 13 0.154 0,077 12 40 12 0.75 0.5 12 fifty 9 0.444 0 12 60 5 0.4 0 12 70 7 0.286 0.286 12 80 3 0.333 0.333 12 90 four 0.25 0 12 one hundred 2 one 0.5 13 twenty one one 0 13 thirty four 0.25 0 13 40 10 0.7 0.7 13 fifty 7 0.429 0.286 13 60 9 0.444 0 13 70 8 0.375 0 13 80 9 0.222 0,111 13 90 7 0.429 0.429 13 one hundred eleven 0.364 0.364 13 110 eleven 0.455 0.273 13 120 one 0 0 fourteen twenty one 0 0 fourteen thirty 7 0.571 0.571 fourteen 40 8 0.5 0.5 fourteen fifty 8 0.125 0.125 fourteen 60 9 0.444 0.222 fourteen 70 6 0.333 0 fourteen 80 8 0.125 0 fourteen 90 7 0.286 0 fourteen one hundred 6 0.5 0.5 fourteen 110 2 0 0 fifteen thirty 5 0.4 0 fifteen 40 5 0.6 0.4 fifteen fifty 3 0 0 fifteen 60 6 0.333 0.333 fifteen 70 5 0 0 fifteen 80 5 0.6 0.6 fifteen 90 7 0.286 0.286 fifteen one hundred 5 0.2 0 fifteen 110 one one 0

Chromosome = number of the chromosome in question; Compartments = specific interval with the number given for the upper limit of the interval of 10 Mbp from the centromere; Frequency = number of SNP markers for each combination of the chromosome and the interval in question; The remaining columns are intended for the proportion of heterozygosity for each combination of the chromosome and the interval in question; chromosome =., compartments =.

Line: General summary for all 1459 markers in the sample; chromosome =., compartments = ** Lines: summary from interval to interval, summarized along all chromosomes; chromosome = **, compartments =.

Lines: summary from chromosome to chromosome, summarized along all intervals; chromosome = **, compartments = **. Lines: A summary for each combination of chromosome and interval.

In all 7 pairs, stem cells showed smaller fractions of heterozygosity than cells of their respective donors. In general, donor cells do not appear to exhibit a clear pattern of a tendency toward heterozygosity along distances from centromeres, while stem cells exhibit slightly smaller portions of heterozygosity near centromeres and telomeres compared to fractions of heterozygosity in the middle.

Based on the results of HLA typing, stem cells obtained from all phESC lines turned out to be MHC-compatible with oocyte donors, which makes this a possible way to create cells for therapeutic use (table 3). HLA analysis of genetic material from human fibroblasts, which were used as nourishing cells, did not reveal phESC line contamination with material from human fibroblasts (table 3).

Table 3
HLA typing results Mhci Mhch Hla-a Hla-b Hla-c DRB1 DQB1 DQA1 phESC-1 A * 01
A * 02 B * 15 (63) B * 35 Cw * 04 Cw * 0708 DRB1 * 12 DRB1 * 13 DQB1 * 06 DQB1 * 03 DQA1 * 01 DQA1 * 0505 phESC-1 donor A * 01
A * 02 B * 15 (63) B * 35 Cw * 04 Cw * 0708 DRB1 * 12 DRBP * 13 DQB1 * 06 DQB1 * 03 DQA1 * 01 DQA1 * 0505 phESC-3,
4, 5 A * 02
A * 03 B * 52
B * 22 Cw * 03 Cw * 04 DRB1 * 01 DRB1 * 03 DQB1 * 05 DQB1 * 02 DQA1 * 0101 DQA1 * 05 phESC-3, 4.5 donor A * 02
A * 03 B * 52
B * 22 Cw * 03 Cw * 04 DRB1 * 01 DRB1 * 03 DQB1 * 05 DQB1 * 02 DQA1 * 0101 DQA1 * 05 phESC-6 A * 02
A * 03 B * 07
B * 27 Cw * 04 Cw * 07 DRB1 * 04 DRB1 * 15 DQB1 * 06 DQB1 * 03 DQA1 * 01 DQA1 * 03 phESC-6 donor A * 02
A * 03 B * 07
B * 27 Cw * 04 Cw * 07 DRBP * 04 DRB1 * 15 DQB1 * 06 DQB1 * 03 DQA1 * 01 DQA1 * 03 phESC-7 A * 01
A * 02 B * 38
B * 57 Cw * 06 Cw * 12 DRB1 * 13 DRB1 * 14 DQB1 * 06 DQB1 * 06 DQA1 * 0106 DQA1 * 0103 phESC-7 donor A * 01
A * 02 B * 38
B * 57 Cw * 06 Cw * 12 DRB1 * 13 DRB1 * 14 DQB1 * 06 DQB1 * 06 DQA1 * 0106 DQA1 * 0103 NSF A * 25
A * 32 B * 15 (62) B * 18 Cw * 12 Cw * 12 DRB1 * 04 DRB1 * 15 DQB1 * 06 DQBl * O3 DQA1 * 01 DQA1 * 03

Analysis of Imprinted Genes

Changes in genomic imprinting in human embryos can contribute to the development of disorders linked to genes expressed in the maternal or paternal organism (Gabriel et al., Proc. Natl. Acad. Sci. USA (1998) 95: 14857-14862). Imprinting studies in phESC require detailed study due to the possible effect on the differentiation of phESC and the functionality of their derivatives. As a preliminary study, the expression of imprinted human genes (Morison et al., Trends Genet. (2005) 21: 457-465) TSSC5, H19, PEG1 and SNRPN in undifferentiated phESC was analyzed (Figure 6). Two hESC lines obtained from unnecessary IVF embryos were used as controls. Transcripts of the maternally expressed TSSC5 genes (Morison et al., 2005, supra) and H19 (Morison et al., 2005, supra) were observed in all phESC lines, as well as in control lines. The human PEG1 gene is transcribed from two alternative promoters (Li et al., 2002, supra). A region of a gene from the first promoter is expressed biallene, and a region of a gene from the second promoter (isoform 1) is expressed paternally (Li et al., 2002, supra). PEG1 gene expression from the first promoter was not affected in phESC lines. Analysis of the paternally expressed region of the PEG1 gene and the paternally expressed SNRPN gene (Morison et al., 2005, supra) showed that the expression of these genes was significantly regulated downward in the phESC lines compared to the hESC control lines (Fig. 6 FP). These results provide additional evidence of the parthenogenetic origin of the described phESC lines.

Example 2. The origin of the line hpSC-Hhom from the HLA-homozygous donor

For the initial purpose of isolating an HLA-homozygous parthenogenetic human stem cell line, oocytes from an HLA-homozygous donor were used. HLA genotyping of the donor (donor 1) and her parents showed that both parents were heterozygous. The same haplotype A * 25, B * 18, DRB1 * 15 was inherited from each parent, and the donor had the HLA homozygous genotype A * 25, A * 25, B * 18, B * 18, DRB1 * 15, DRB1 * 15 (table 5, case 1).

Nineteen cell-oocyte complexes (SOS) were taken from donor 1, of which seven were used for research (table 4).

Table 4
The origin of parthenotes and HLA-homozygous human stem cell lines Donor number Collected oocytes Donated Oocytes Blastocysts Cell lines IVF result one 19 7 four hpSC-Hhom-1 Successfully 2 eighteen 7 3 hpSC-Hhom-2 hpSC-Hhom-3 Successful (twin pregnancy) 3 twenty 10 0 0 To no avail four 27 fourteen 2 hpSC-Hhom-4 Successfully Total 84 38 9 four Data not available

Parthenogenetic activation was performed using the previously described protocol with A23187 and 6-DMAP processing (Revazova et al., 2007, supra). Four parthenogenetic embryos reached the blastocyst stage, from which the hpSC-Hhom-1 line was isolated from one.

Genotypic relationships between hpSC-Hhom-1 cells and somatic donor 1 cells were identified as “full siblings” (genetic matching) using SNP analysis. Comparison of SNP markers showed that donor 1 cells had a heterozygosity pattern, while hpSC-Hhom-1 cells showed a smaller proportion of heterozygosity.

HLA genotyping showed that the hpSC-Hhom-1 line is HLA homozygous: A * 25, A * 25, B * 18, B * 18, DRB1 * 15, DRB1 * 15 and is completely HLA-compatible with the donor (table 5, case 1).

Table 5
HLA genotyping Happening Hla-a Hla-b DRB1 one Donor mother A * 25
A * 26 B * 18
B * 44 DRB1 * 15
DRB1 * 11 Donor's father A * 25
A * 03 B * 18
B * 07 DRB1 * 15
DRB1 * 15 Donor A * 25
A * 25 B * 18
B * 18 DRB1 * 15
DRB1 * 15 hpSC-Hhom-1 A * 25
A * 25 B * 18
B * 18 DRB1 * 15
DRB1 * 15 2 Donor mother A * 02
A * 33 B * 13
B * 14 DRB1 * 07
DRB1 * 01 Donor's father A * 68
A * 03 B * 18
B * 40 DRB1 * 13
DRB1 * 03 Donor A * 02
A * 68 B * 13
B * 18 DRB1 * 07
DRB1 * 13 hpSC-Hhom-2 A * 68
A * 68 B * 18
B * 18 DRB1 * 13
DRB1 * 13 hpSC-Hhom-3 A * 02
A * 02 B * 13
B * 13 DRB1 * 07
DRB1 * 07 3 Donor mother A * 02
A * 01 B * 08
B * 15 DRB1 * 03
DRB1 * 01 Donor's father A * 01
A * 33 B * 51
B * 15 DRB1 * 11
DRB1 * 04 Donor A * 02
A * 01 B * 08
B * 51 DRB1 * 03
DRB1 * 11 HLA haplotype N10: A * 02 B * 08 DRB1 * 03 no line four Donor mother A * 02
A * 02 B * 07
B * 40 DRB1 * 13
DRB1 * 16 Donor's father A * 01
A * 24 B * 08
B * 44 DRB1 * 03
DRB1 * 01 Donor A * 02
A * 01 B * 40
B * 08 DRB1 * 13
DRB1 * 03 hpSC-Hhom -4 A * 01
A * 01 B * 08
B * 08 DRB1 * 03
DRB1 * 03 HLA haplotype N1 Nourishing cells NSF A * 25
A * 32 B * 15 (62)
B * 18 DRB1 * 04
DRB1 * 15

Bold HLA haplotype of the mother of the donor; underlined is the HLA haplotype of the donor's father, each of which was inherited by the donor, and subsequently by the hpSC-Hhom line.

The origin of the hpSC-Hhom lines from an HLA heterozygous donor

Since HLA-homozygous oocyte donors are rare, resorted to the isolation of HLA-homozygous cell lines from oocytes obtained from HLA-heterozygous donors. A total of eighteen SOS were received from donor 2, of which seven were donated for research (Table 4). These oocytes were parthenogenetically activated using another protocol treated with A23187 and puromycin. After 18 hours, the ejection of the 2nd polar body and the formation of one pronucleus in activated oocytes were observed. Three blastocysts developed from these zygotes, which allowed us to distinguish two hpSC-Hhom lines: hpSC-Hhom-2 and hpSC-Hhom-3.

SNP analysis performed between the somatic cells of the oocyte donor 2 and the hpSC-Hhom-2 and hpSC-Hhom-3 lines showed a parent-child relationship. Moreover, both of these cell lines turned out to be homozygous throughout the genome (score by SNP markers), as opposed to heterozygous somatic donor cells.

Based on the results of HLA genotyping, cells from both of these lines turned out to be HLA homozygous: the hpSC-Hhom-2 line showed the HLA genotype A * 68, A * 68, B * 18, B * 18, DRB1 * 13, DRB1 * 13, and the hpSC-Hhom-3 line showed the HLA genotype A * 02, A * 02, B * 13, B * 13, DRB1 * 07, DRB1 * 07 (at the studied loci). Each hpSC-Hhom line also inherited a different HLA haplotype, one from the donor’s father and the other from the donor’s mother. The HLA haplotype of the donor father (A * 68, B * 18, DRB1 * 13) was found in the homozygous state in the hpSC-Hhom-2 line, and the HLA haplotype of the donor mother (A * 02, B * 13, DRB1 * 07) was found in a homozygous state in the hpSC-Hhom-3 line (table 5, case 2).

Isolation of hpSC-Hhom lines from oocytes of HLA-heterozygous donors selected by the HLA haplotype

As a final step, hpSC-Hhom lines with the HLA haplotype, which is known to occur with a high frequency in the population, were isolated. Screening for IVF candidates for the HLA haplotype yielded two HLA heterozygous oocyte donors bearing a common haplotype.

According to the published frequency list of the HLA haplotype (Mori et al., Transplantation (1997) 64: 1017-1027), donor 3 (HLA haplotype A * 02, B * 08, DRB1 * 03) and donor 4 (HLA haplotype A * 01, B * 08, DRB1 * 03) (Table 5, Case 3 and Case 4) carried the haplotypes common in the US population. However, with the heterozygous HLA genotype, each donor carried not only a frequently occurring haplotype, but also a less common one. Thus, it was impossible to predict with absolute accuracy which haplotype would be present in the selected hpSC-Hhom line.

A23187 and puromycin were used for parthenogenetic activation of donor oocytes. Isolation of the hpSC-Hhom line from donor 3 oocytes was not successful, from which twenty SOS were obtained and 10 oocytes were donated for the study. None of them reached the blastocyst stage. Moreover, the IVF procedure in this donor did not lead to pregnancy. Together, these facts may reflect the poor quality of the oocytes from this particular donor (table 4).

27 SOS was received from donor 4, and fourteen oocytes were donated for the study. After parthenogenetic activation of oocytes, two blastocysts were obtained, from which the hpSC-Hhom-4 line was isolated (table 4).

The genotypic relationship between hpSC-Hhom-4 and somatic cells of donor 4 was identified as a “parent / offspring pair” using an SNP analysis similar to case 2. The hpSC-Hhom-4 line was homozygous for the whole genome (estimated by SNP markers), compared with heterozygous somatic donor cells (1,174 heterozygous SNP markers).

According to HLA genotyping, the hpSC-Hhom-4 line was HLA-homozygous (at the studied loci) and had the most common haplotype in the USA, common for a number of racial groups (Table 6): A * 01, B * 08, DRB1 * 03 ( table 5, case 4).

Table 6
Frequency and ranking according to the racial group for the HLA haplotype A * 01, B * 08, DRB1 * 03 in the US population
(adapted from Mori et al., 1997, above) Racial group ^a Frequency (%) ^b Ranking ^with CAU 5.1812 one NAT 4.7439 one AFR 1.2491 2 Lat 1.6733 3 ASI 0.3195 54 ^and White Americans (CAU), Native Americans (NAT), African Americans (AFR), Hispanics (LAT), and Asian Americans (ASI).
^b frequency of haplotypes HLA-A, -B, -DR
^with appropriate ranking within each racial group

The IVF procedure resulted in pregnancy in three of four donors (donors 1, 2, and 4). A large proportion of successful IVFs were largely due to the selection of donors with a good prognosis for pregnancy after IVF. Interestingly, two hpSC-Hhom lines were isolated from donor 2, and after the IVF procedure, the donor became pregnant with twins.

Characterization of hpSC-Hhom lines

Cells from all hpSC-Hhom lines showed the morphology that was expected from human embryonic stem cells, formed colonies from tightly packed cells, and showed prominent nucleoli at a low ratio of cytoplasm and nucleus. All cells expressed common human embryonic stem cell markers SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and OST-4 and did not express SSEA-1, a positive marker for undifferentiated murine embryonic stem cells (Figure 7 ) All lines showed high levels of alkaline phosphatase activity (Figure 7) and telomerase (Figure 8).

Giemsa disk karyotyping showed that the hpSC-Hhom-1 and hpSC-Hhom-4 lines have a normal human 46, XX karyotype (Figure 9, A and B). The hpSC-Hhom-2 and hpSC-Hhom-3 lines obtained from the same donor had deviations in the karyotype. Approximately 15% of cells from the hpSC-Hhom-2 line have chromosome 8 aneuploidy: karyotype 47, XX, +8 (Figure 9, C), and 4.2% of the hpSC-Hhom-3 cells have chromosome 1 aneuploidy: karyotype 47 XX, +1 (Figure 9, D). Heteromorphism of the X chromosome was not observed in any cell line when analyzing 100 metaphases (figure 9).

The hpSC-Hhom-4 line remained undifferentiated for 27 passages. Other cell lines were successfully cultured for at least 21 passages. Cells from the hpSC-Hhom-4 line formed cystic embryoid bodies in suspension culture and gave rise to derivatives of all three germ layers — ectoderm, mesoderm, and endoderm — after differentiation in vitro (Figure 10). Ectodermal differentiation was confirmed by positive immunocytochemical staining for neuron-specific markers neurofilament 68 (Figure 10A) and NCAM (Figure 10B). Differentiated cells were also positive for mesodermal markers, including muscle-specific markers desmin (Figure 10C) and alpha-actinin (Figure 10D). Endodermal differentiation was confirmed by positive staining for alpha-fetoprotein (Figure 10E).

The ability of all hpSC-Hhom lines to form derivatives of all three germ layers was further studied in vivo by subcutaneous injection of hpSC-Hhom cells in immunodeficient mice (Figure 11). All hpSC-Hhom lines were able to form teratomas approximately two months after injection. Teratocarcinomas were not observed. Approximately four million mitomycin-C-treated human fibroblasts used as feed layers for hpSC-Hhom cells were also injected as controls, and teratoma growth was not observed.

Histological examination of cell transplants revealed organized structures, including various types of glands (some produced brown pigment, possibly bile pigment), cartilage differentiation, well-formed bones, mesenchymal cells, high production of collagen fibers, adipose tissue, neural tubes, and multilayered lining epithelium with pearls of parakeratosis (figure 11). These data suggest that hpSC-Hhom will differentiate in vivo in tissues originating from all three germ layers.

Although the invention is described with reference to the above examples, it should be understood that its modifications and variations are included in the scope and covered by the idea of the invention. Accordingly, the invention is limited only by the following claims.

Claims (13)

1. The method of generating a selected line of human stem cells derived from parthenogenetic blastocysts, including:
a) incubation of unfertilized human oocytes in metaphase II in in vitro fertilization media (IVF), where the oocytes do not have paternal imprinting and do not have foreign DNA;
b) incubation of cells of stage (a) in IVF media, including ionophore;
c) incubating the cells of step (b) in IVF media including puromycin; and
d) incubation of the cells of stage (C) in fresh IVF medium at a low partial pressure of O ₂ ,
in which the incubation stages (a) to (c) are carried out at a high partial pressure of O ₂ , where the high partial pressure of O ₂ is about 20% O ₂ and the low partial pressure of O ₂ is about 5% O ₂ , where the internal cell masses (ICM ) obtained from the cells in step (c) produce stem cells suitable for cultivation and where
at least one cell constituting the cell line is homozygous throughout the genome according to SNP markers relative to donor heterozygous somatic cells. 2. The method according to claim 1, where at least one cell forms a teratoma after transplantation of a immunocompromised mouse. 3. The method according to claim 1, where at least one cell is MHC-compatible with an unfertilized oocyte donor. 4. The method according to claim 3, where at least one cell is MHC compatible with a direct relative of the blastocyst donor. 5. The method according to claim 1, where at least one cell has undergone significant genetic imprinting in accordance with the origin of the donor. 6. The method according to claim 1, where at least one cell (i) will proliferate in an in vitro culture for more than one year, (ii) retains the ability to differentiate into derivatives of endoderm, mesoderm and ectoderm tissues throughout the culture and (iii) amenable to inhibition of differentiation when it is cultivated on the feeding sub-layer of fibroblasts. 7. The method according to claim 6, where at least one cell differentiates into a cell selected from the group consisting of a neuronal cell, cardiomyocyte, smooth muscle cell, striated muscle cell, endothelial cell, osteoblast, oligodendrocyte, hematopoietic cell, fat cell , stromal cell, chondrocyte, astrocyte, keratinocyte, pancreatic islet cell, lymphoid progenitor cell, mast cell, mesoderm cell and endoderm cell. 8. The method according to claim 1, where the cell line is obtained from parthenogenetic blastocysts, which does not have the ability to form a viable organism. 9. The method according to claim 1, in which at least one cell expresses one or more markers selected from the group consisting of SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and OST-4 . 10. A method of treating a subject in need thereof, comprising administering a cell composition comprising differentiated cells, wherein the differentiated cells are derived from a stem cell line obtained according to the method of claim 1. 11. The method according to claim 10, in which the subject has a disease selected from the group consisting of Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS, defects or injuries of the spinal cord, multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, macular degeneration, defects or damage to cartilage, burns, foot ulcers, vascular disease, urinary tract disease, AIDS and cancer. 12. A method for generating HLA-homozygous stem cells, comprising:
a) screening of oocyte donors for HLA haplotypes, widespread in this population group;
b) incubation of unfertilized human oocytes in metaphase II in in vitro fertilization media (IVF), where the oocytes do not have paternal imprinting and do not have foreign DNA;
c) incubating the cells of step (b) in IVF media including an ionophore;
d) incubating the cells of step (c) in IVF media including puromycin; and
e) incubating the cells of stage (d) in fresh IVF medium at a low partial pressure of O ₂ ,
wherein the incubation steps (b) to (d) are carried out at a high partial pressure of O ₂ , where the high partial pressure of O ₂ is about 20% O ₂ and the low partial pressure of O ₂ is about 5% O ₂ , and in which the internal cell masses (ICM) obtained from cells in step (d) produce cultured stem cells. 13. The method of claim 12, further comprising HLA typing of the biological parents of the oocyte donor of stage (a).

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